Methods and Compositions Comprising Capture Agents

ABSTRACT

The present invention relates to methods and compositions for detecting a substance using mixed or multiple element capture agents (MECA). The affinity of a MECA for a target is produced by the concomitant binding of at least two low to moderate affinity capture agents providing a high affinity interaction with a capture target.

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/438,805, filed on Jan. 9, 2003, which is incorporated hereinby reference in its entirety.

The government has rights in the present invention pursuant to grantnumber R21CA932701 and R21CA093287 from the National Cancer Institute,contract No. NO1-HV-28185 from the National Institutes of Health, andgrant number 1-1299 from the Welch Foundation.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of chemistry,molecular biology, and diagnostics. More particularly, it concernsmethods and compositions for obtaining a high affinity synthetic captureagent for a molecular or biomolecular target by co-immobilizing at leasttwo low-to-moderate affinity ligands on a suitable surface. Such a highaffinity synthetic capture agent is referred to as a mixed elementcapture agent (MECA). MECAs will be of great utility in the constructionof medical diagnostic devices.

2. Description of Related Art

There is great interest in the development of techniques with which tomonitor the levels, post-translational modification states andactivities of large numbers of proteins simultaneously. One approach isto construct protein-detecting arrays (Kodadek, 2001; Kodadek, 2002),akin to the DNA microarrays used widely in genomics research. Suchdevices may be comprised of many different protein-binding moleculesarrayed on a suitable surface in a defined pattern, each capable ofrecognizing its target protein with high affinity and high specificity.These high affinity and high specificity protein-binding molecules orligands are referred to as capture agents. An alternative format is toimmobilize protein-binding molecules on encoded beads (Vignali, 2000;Oliver et al., 1998). A significant challenge in the development of suchtechnology is the isolation of large numbers of protein-bindingcompounds with a sufficiently high binding affinity and specificity tobe useful in the capture of particular proteins from a complex mixture.

Most of the effort in this area has focused on the use of macromolecularbiomolecules as capture agents, such as antibodies (Eggers et al., 1998;Hunag et al., 2002; Huang, 2001; Wiese et al., 2001; Huang et al., 2001;Walter et al., 2000; and Haab et al., 2001), nucleic acids (particularlyRNA) aptamers (Osborne et al., 1997; Jhaveri et al., 2000; Famlouk andJenne, 1998; Seethsnunan et al., 2001; and Vaish et al., 2002) andprotein-RNA fusions (Roberts and Szostak, 1997; Wilson et al., 2001; andColas et al., 1996).

Protein-binding molecules can be readily isolated from combinatoriallibraries or other types of large compound collections using a number ofmethods. Unfortunately, small molecules, peptides, peptidomimetics andother synthetically accessible compounds rarely bind to their targetprotein with an affinity comparable to that of a good antibody(equilibrium dissociation constant (K_(D))≦10⁻⁹ M). Instead, smallmolecule/protein complexes generally exhibit K_(D)s in the μM range,with the exception of molecules optimized through extensive medicinalchemistry efforts or natural selection. This modest affinity isinsufficient to capture low abundance proteins from complex mixtures. Inaddition, the relatively rapid dissociation rates of such complexesresult in the loss of most of the bound capture target during theinevitable washing steps required to minimize non-specific “background”binding of high abundance or “sticky” proteins. Therefore, a centralproblem in applying organic chemistry to the development ofprotein-detecting microarrays is obtaining higher affinity syntheticligands in a high-throughput fashion.

In the case of pharmaceuticals, optimization of a lead compound isgenerally achieved through a tedious and labor-intensive process inwhich hundreds of relatives of the lead molecule are synthesized andevaluated for activity. It is not possible to apply this approach on ascale where ligands are required for hundreds or even thousands ofproteins. One potential “shortcut” in the path from low to high affinityagents is to employ multivalency. For example, coupling two or moremodest affinity protein ligands with an appropriate linker can provide ahigh affinity bivalent or multivalent ligand (Shukery et al., 1996;Olejniczak et al., 1997; Thorn et al., 2001; Terskikh et al., 1997;Merritt et al., 2002; Kitov et al., 2000; Kiessling et al., 2000 andCussac et al., 1999). Unfortunately, linker optimization can be timeconsuming, and most approaches to this problem are unsuitable forhigh-throughput proteomics applications (see Maly et al., 2000, for anexemplary combinatorial approach). Thus, there remains a need for rapid,selective and high-affinity compositions and methods for rapidlyproviding a high affinity synthetic capture agent.

SUMMARY OF THE INVENTION

This invention describes how two low to modest affinity binding elementscan be combined on a surface in such a way as to form a high affinitycapture agent without any requirement for the design or discovery of asuitable linker to connect the two ligands. Various embodiments of theinvention include novel capture agent compositions and methods forobtaining these types of mixed element capture agents (MECAs) even ifonly a single binding element is known for a given molecular target. Incertain embodiments, methods and compositions of the invention include aplurality of low to moderate affinity binding elements distributed on asurface of and operatively coupled to a support, wherein concomitantbinding of a first target molecule to two of the low-to-moderateaffinity binding elements results in a high affinity interaction withthe first target molecule producing a mixed or multiple element captureagent (MECA).

In certain embodiments, MECAs are comprised of two or more bindingelements that have been previously demonstrated to bind to the targetmolecule, for example a protein, with low to modest affinity (defined asan equilibrium dissociation constant (K_(D)) between 10⁻³ M and 10⁻⁸ M).The other stipulation is that these binding elements do not compete forbinding to the target molecule. In certain embodiments, these low tomodest affinity binding elements may be peptides, peptoids or otherpeptide-like oligomers. The crux of the invention is that if two or moresuch low-to-moderate affinity binding elements are immobilized(covalently or non-covalently) at high density on a suitable surface,then some fraction of the possible pairs of molecules on the surfacewill have an appropriate geometry relative to one another to bind thetarget molecule cooperatively. In other words, the surface will providea “library” of suitable linker geometries. This allows for the creationof a bidentate (or multidentate) capture agent that will retain thetarget molecule with high affinity and specificity without any effortexpended in linker discovery, design and optimization.

The individual binding elements that make up a MECA may be combined onthe surface in a number of ways. Either two or more binding elements canbe added simultaneously to a suitably functionalized surface in order toachieve a statistical distribution of the two or more binding elementson the surface, or two or more binding elements may be operativelycoupled to one another prior to immobilization. In the latter case, asingle chimeric species, containing two binding elements, would then beattached to the surface, providing the highest possible density ofimmobilized species. This pre-linkage could consist of linear orbranched fusions of the ligands or coupling them to a variety ofscaffolds. Importantly, these couplings would not involve any linkerdesign, discovery or optimization. In various embodiments of theinvention, a support is a cross-linked polymer bead or achemically-modified glass slide. Samples that could be analyzed withthese immobilized capture agents include, but are not limited to, a celllysate, a blood sample, a sputum sample, or a urine sample. Samples mayinclude various other biological and non-biological materials. A targetmolecule may be any molecule or substance to which a MEGA may bindincluding, but not limited to, polypeptides. Targets could also includeassemblages of molecules, such as multi-protein complexes, sub-cellularcompartments (nucleus, etc.) or even whole cells. A target polypeptidemay also be a proteolytic fragment or part of polypeptide or may carrypost-translational modifications. Post-translational modificationincludes, but is not limited to phosphorylation, glycosylation,ubiquitylation, SUMOylation (Sternsdorf et al., 1999) and the like.

In various embodiments, compositions may comprise a third and a fourthlow-to-moderate affinity binding element that bind a second targetmolecule (i.e., a second MECA), the third and fourth low affinitybinding element are distributed on a surface of, and operatively coupledto, a support, wherein concomitant binding of the second target moleculeto the third and fourth low affinity binding elements results in a highaffinity interaction with the second target molecule. The third andfourth low affinity binding elements typically have a distinct bindingspecificity as compared to each other. The third and fourth low tomoderate affinity binding elements (second MECA) will typically havedistinct binding specificity as compared to a first and second lowaffinity binding elements (first MECA). In other words, the second MECAwill capture a different target molecule or assemblage of molecules thanthe first MECA. In various embodiments, a composition may include 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 100, 1000 or more MECAs that can be arranged inan addressable array, wherein each MECA or a group of MECAs may besegregated from other MECAs or groups of MECAs.

Once a target molecule or assemblage of molecules is captured by a MECA(or multiple targets are captured on an array of MECAs), these bindingevents can be detected and quantified by a variety of methods including,but not limited to spectroscopy, mass spectrometry, fluorescence,magnetic resonance imaging surface plasmon resonance and the like.

In certain embodiments, contemplated methods include analyzing themodification of a protein in a sample. The method may include exposingthe protein-containing sample to one or more MECAs, which would bedesigned to discriminate between different modified forms of theprotein. This would be achieved by co-immobilizing one or morelow-to-modest affinity binding elements that recognize the protein andwhich are relatively insensitive to the modification, with one or morelow-to-modest affinity binding elements that recognize the modification(for example a ubiquitin molecule or phosphoserine) but which isrelatively insensitive to the identify of the polypeptide. This type ofMECA would have a high affinity for only the protein of interestdecorated with the post-translational modification of interest, whereasother proteins modified in this way or the target protein decorated withother types of modifications would evince only low-to modest affinitybinding to the MECA.

In another embodiment, a specific multi-protein complex could becaptured by a MECA comprised of ligands for two or more differentproteins in the complex.

Another embodiment of the invention includes methods of discoveringMECAs when only a single low-to-modest affinity ligand for a givenmolecular target is available. This is done by coupling the knownlow-to-moderate affinity binding element to each molecule in acombinatorial library of oligomers. The library is screened on asuitable surface (modified polystyrene bead, chemically modified glassslide, etc.) under conditions that demand a high affinity capture eventto retain the target protein on the surface. This procedure will allowthe high affinity, cooperative binding of the lead compound and alibrary-derived molecule to the target molecule. Capture agents derivedvia this method are described below as chimeric mixed element captureagents. The oligomer may be a peptide or peptide derivative, nucleicacid or nucleic acid derivative, or an oligomer comprising a pluralityof heterogeneous monomer units. A peptide derivative may include one ormore non-natural amino acids and may include one or more peptoidmonomers. In certain aspects, the binding element or ligand may be anucleic acid, amino acid, peptide, steroid, inorganic molecule ororganic molecule. The ligand may be operatively coupled to a terminal orinternal position in an oligomer.

Certain embodiments include compositions for assessing the presence ofat least a first target molecule in a sample comprising a plurality oflow-to-moderate affinity binding elements distributed on a surface of,and operatively coupled to a support, wherein concomitant binding of thefirst target molecule to two or more of the binding elements results ina high affinity interaction with the first target molecule. In certainaspects the binding element are known low to moderate affinity bindingelements of a target molecule. The binding elements may be, but are notlimited to peptides, peptoids (N-substituted oligoglycines) or otherpeptide-like oligomers. In further embodiments, the plurality of bindingelements comprises at least a first and a second binding element havingdistinct binding specificity for a target molecule as compared to eachother. The first binding element may be operatively coupled to thesecond binding element. A spacer may be operatively coupled to the firstbinding element, the second binding element or both the first and secondbinding element. The second binding element may be an oligomer. Theoligomer may be, but is not limited to a peptide or peptide derivative.A peptide derivative or a peptide like molecule is comprised of one ormore non-natural amino acid or analogous molecule. The peptidederivative may be comprised of one or more peptoid monomers. A firstbinding element may be a nucleic acid, peptide, steroid, inorganicmolecule or organic molecule. In certain embodiments, one or more firstbinding element may be operatively coupled to a terminal and/or internalmonomer of the oligomer or second binding element.

In still further embodiments of the invention a sample may be anenvironmental sample, a cell lysate, a blood sample, a sputum sample ora urine sample. The sample may include one or more target molecules. Atarget molecule may a biological molecule or metabolite. A biologicalmolecule is molecule produced or utilized by an organism. An organismincludes, but is not limited to humans, mammals, pathogens, microbes,bacteria, fungi, virus, prokaryotes and eukaryotes. The target moleculemay be a polypeptide. The polypeptide may be modified. Modificationincludes, but is not limited to phosphorylation, SUMOylation orubiquitylation. A target molecule may or may not be coupled to adetectable label.

The binding elements are typically distributed randomly on the surfaceof the support. A support may be a cross-linked polymer bead or achemically-modified glass slide.

In still further embodiments, the composition may include at least athird and a fourth low-to-moderate binding element that bind a secondtarget molecule, the third and fourth binding element distributed on asurface of, and operatively coupled to, the support, wherein concomitantbinding of the second target molecule to the third and fourth bindingelements results in a high affinity interaction with the second targetmolecule. The third and fourth low affinity binding elements may havedistinct binding specificity as compared to each other and/or to thefirst and second low affinity binding elements. The first and second lowaffinity binding elements may be segregated from at least the third andfourth low affinity binding elements. In certain embodiments, the firstand second low affinity binding elements are segregated from the thirdand fourth low affinity binding elements on the surface of the support.The first and second binding elements, and the third and fourth bindingelements, are typically distributed randomly on the surface of thesupport within their respective segregated areas.

In other embodiments, methods of determining the presence of targetmolecule in a sample are contemplated that comprise a) exposing thesample to a plurality of low-to-moderate affinity binding elementsdistributed on a surface of, and operatively coupled to a support,wherein concomitant binding of the target molecule to at least a two ofthe binding elements results in a specific high affinity interactionwith the target molecule; and b) evaluating binding of the targetmolecule to the binding elements. Binding may be observed byspectroscopy. Spectroscopy may be fluorescent and/or magnetic resonanceimaging spectroscopy. The binding of a protein may be compared with thebinding of the unmodified protein.

Embodiments of the invention include methods of producing a chimericbinding element comprising a) providing a first low-to-moderate affinitybinding element; b) providing a combinatorial library of oligomers; c)operatively coupling the first binding element to one or more members ofthe combinatorial library; and d) identifying a first bindingelement/oligomer combination with a high affinity for a target molecule,wherein at least a portion of the oligomer is a second binding element.The oligomer may be a peptide or peptide derivative. A peptidederivative is typically comprised of one or more non-natural amino acidor analogous molecule. The peptide derivative may comprise one or morepeptoid monomers.

In certain embodiments, a composition for assessing the presence of atleast a first target molecule in a sample comprising chimeric bindingelements distributed on a surface of, and operatively coupled to asupport, wherein concomitant binding of the first target molecule to twoor more of the chimeric binding elements results in a high affinityinteraction with the first target molecule is contemplated.

It is contemplated that any embodied method or composition describedherein can be implemented with respect to any other method orcomposition described herein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 is a schematic diagram of one anticipated mode of binding of adimeric protein with a surface densely functionalized with a captureagent. Some fraction of the molecule pairs on the surface areanticipated to have the correct spacing to facilitate high-affinity,bidentate binding, as represented by length “a”. Distances betweenmolecules other than length “a,” denoted as “b,” “c,” and “d,” will notsupport bidentate binding.

FIG. 2 illustrates an example of high affinity protein ligand created byimmobilizing two different modest affinity binding elements onto asurface. The surface is anticipated to act as a “library of linkers.” Inother words, the two binding elements should be spaced appropriately onsome fraction of the surface to bind in a bivalent fashion to theprotein.

FIGS. 3A-3D illustrate examples of different ways in which modestaffinity binding elements could collaborate when immobilized to asupport to capture proteins with high affinity.

FIG. 4 illustrates an example of how high affinity binding of aparticular post-translationally modified protein through a two-pointcontact is accomplished.

FIG. 5 illustrates an exemplary characterization of Gal80-bindingpeptides isolated by phage display. Gal80 is a homodimer that can alsotetramerize at concentrations above 100 nM. Note that the solutionbinding curve shown on the left reveals that the K_(D) of thepeptide-protein complex in solution is modest 0.3

FIGS. 6A-6B illustrate an example of slow dissociation of Gal80 proteinfrom Tentagel-bound binding peptide. FIG. 6A illustrates a schematicrepresentation of the assay employed to monitor the half-life of theimmobilized peptide/protein complexes. FIG. 6B shows SDS-PAGE/Westernblot analysis of the amount of Gal80 remaining on the peptide-coatedbeads following dilution. Time elapsed following dilution is indicatedin part A for each tube. Lanes 10 contain samples in which thepeptide-coated beads were added to the highly dilute protein solution(approx. 5 pmol) and incubated for two hours to control for proteinreassociation with the beads under these conditions. Lanes 11 representexperiments using scrambled sequences attached to the beads. Lanes 12represent experiments done with Tentagel beads lacking any peptide. Notethat dissociation of the peptide/protein complex in solution occurredwithin seconds (data not shown).

FIGS. 7A-7B illustrate an example of efficient capture of a dilutedimeric protein by immobilized peptides. (FIG. 7A) The amount ofHis₆Gal80 protein indicated was added to 500 μl of buffer containing 5.7mg E. coli lysate and 2 mg of Tentagel-Gal80 bp. After incubation andwashing, the amount of protein retained on the beads is shown. (FIG. 7B)Calibration blot using known amounts of purified His₆Gal80 protein. Thisfigure shows that the immobilized peptide binds the Gal80 dimer with asub-nanomolar dissociation constant.

FIGS. 8A-8B are an exemplary comparisons of the dissociation rates fromimmobilized peptides of monomeric and dimeric fusion constructscontaining the same target protein. A protocol similar to that describedfor FIG. 6A was employed, except fewer time points were taken. (FIG. 8A)SDS-PAGE/western blot analysis showing the levels of dimeric GST-Mdm2 ormonomeric MBP-Mdm2 remaining associated with Tentagel-Mdm2 bp afterdilution for the time indicated. (FIG. 8B) A similar experiment wasconducted with dimeric GST-KIX or monomeric His₆-KIX. Lanes 1 and 3 showthe protein standards. Lanes 2 and 4 show the amount of proteinremaining on the Tentagel-KIXbp1 beads immediately following the washingstep. This experiment proves that the dimmers are held tightly via atwo-point contact and validate the idea that two different bindingelements that bind the same monomeric protein non-competitively couldcollaborate to bind a target protein with very high affinity.

FIGS. 9A-9B illustrate exemplary results from a study using twodifferent binding elements that bind different surfaces of a monomer(i.e., a MBP-Mdm2 fusion protein). A fluorescently labeled fusionprotein was incubated under demanding conditions (high salt anddetergent) with beads that displayed either a MBP-binding peptide, aMdm2-binding peptide, or both (i.e., a MECA comprised of a single linearpeptide in which the two component binding elements were linked via asingle serine residue, i.e. a chimeric binding element). FIG. 9A showsthe beads displaying both peptides bound the Tx Red-labeled fusionprotein to a 1:1 mixture of beads displaying either the simple peptideor MECA. The micrographs show that in each case, the MECA bound thelabeled fusion protein with much higher affinity than the beads thatdisplayed a single peptide. Binding studies (FIG. 9B) done by isothermalcalorimetry revealed that in free solution however, the MECA binds thefusion protein only slightly (<2-fold) better than the componentpeptides, highlighting the importance of surface immobilization in theresult shown in FIG. 9A. Presumably, the spacing between the peptides inthe MECA is inappropriate for high affinity, bidentate 1:1 binding insolution.

FIG. 10 is a schematic model of the presumed mode of binding of atwo-domain protein (such as MBP-mdm2) to a MECA comprised of fused orchimeric binding elements. Note that 1:1 binding would be realized onlyin the unlikely event that the linker provided ideal geometry forbidentate interactions. This can be determined by asking if the solutionK_(D) of the MECA/protein complex is much lower than those of theindividual binding element complexes.

FIG. 11 is a schematic of a model for the rapid isolation of highaffinity capture agents for monomeric proteins. The idea being to takeadvantage of the fact that most monomeric proteins are composed ofseparable, independently expressible domains. Two or more domains wouldthen be combined on the support to provide a MECA for that protein.

FIG. 12 is a list of exemplary amines used for the preparation ofpeptoid libraries. Included in brackets is the correspondingnomenclature of the peptoid units.

FIG. 13 illustrates the text sequences of ten random peptoids pickedfrom the 78,125 compound library. The sequences, were determined byautomated Edman degradation.

FIG. 14 illustrates the fluorescence emission spectrum of a TentaGelbead. Excitation: 460-490 nm. Emission of some fluorescent dyes: (a)Fluorescein, (b) Tetramethylrhodamine, and (c) Texas Red.

FIGS. 15A-15C show isolation of a putative peptoid ligand for MBP-Mdm2.(FIG. 15A) A photomicrograph showing a field of beads that contains theone picked as a putative “hit.” (FIG. 15B) An Edman sequencing trace ofthe bright bead shown in part (FIG. 15A). (FIG. 15C) The sequence of theisolated peptoid deduced from automated Edman degradation from thesingle bead.

FIGS. 16A-16B show characterization of the peptoid/protein complex byisothermal titration calorimetry (ITC). ITC traces for binding ofNlys-Nbsa-Nlys-Nser-Nbsa-Npip-Nbsa-Npip to: (FIG. 16A) MBP-Mdm2 and(FIG. 16B) MBP. The top panel shows the raw data whereas the bottompanel shows the integrated curve of the experimental points (solidcircles) and the best fit (solid line) of the curve. The K_(D) valuesderived from these data were 37 μM for the MBP-Mdm2/peptoid complex andgreater than 1 mM (i.e., little or no binding) for MBP, indicating thatthe peptoid targets the Mdm2-derived polypeptide.

FIGS. 17A-17B show characterization of the on-resin binding propertiesof the Mdm2-binding peptoid. (FIG. 17A) TentaGel beads displayingNlys-Nbsa-Nlys-Nser-Nbsa-Npip-Nbsa-Npip were incubated with 500 nM ofTexas Red-labeled MBP-Mdm2 (left panel) or 500 nM Texas Red-labeled MBP(right panel). (FIG. 17B) Capture of native protein. TentaGel beadsdisplaying the peptoid indicated were incubated with 1 native proteinand a 1000-fold excess of E. coli extract. The protein retained wasanalyzed by SDS-PAGE. A Western blot using anti-Mdm2 antibody is shown.Lane 1: molecular mass standards. Lane 2: 20% of the input. Lane 3:protein retained by TentaGel beads displaying the hitNlys-Nbsa-Nlys-Nser-Nbsa-Npip-Nbsa-Npip. Lane 4: protein retained by thecontrol peptoid Nmba-Nbsa-Nleu-Nlys-Npip-Nmba-Nleu-Nleu. Lane 5: Proteinretained by TentaGel beads lacking a displayed peptoid (bead onlycontrol).

FIGS. 18A-18C show characterization of a large peptoid librarycontaining more than half a million compounds. (FIG. 18A) Sequences ofthe peptoids obtained from ten beads picked randomly from the library.(FIG. 18B) Representative Edman traces obtained from one of these beads.(FIG. 18C) HPLC traces of two hexamers (Ntrp-Nmea-Npip-Nlys-Nffa-Nmbaand Nbsa-Nleu-Napp-Napp-Nffa-Nmea-Npip) that, between them, contain eachof the amines employed in the construction of the library.

FIGS. 19A-19B show identification of a GST-binding peptoid from alibrary of 100,000 pentamers. (FIG. 19A) Edman traces of the hit pickedfrom the screening experiment. (FIG. 19B) Sequence of the peptoidderived from the Edman traces.

FIGS. 20A-20C show characterization of the on-bead binding properties ofthe peptoid obtained in the screen against GST. (FIG. 20A)Photomicrographs obtained after incubation of TentaGel beads displayingthe putative GST-binding peptoid Nbsa-Nlys-Nbsa-Npip-Nlys (left andmiddle panels) or a control peptoid Npip-Nser-Nbsa-Nall-Nlys-Npip (rightpanel) with 500 nM Texas Red-labeled GST or 500 nM Texas Red-labeledMBP. Two percent BSA was included in each solution to reducenon-specific interactions. (FIG. 20B) Capture of native GST byTentaGel-displayed peptoid. A Western blot obtained using anti-GSTantibodies is shown. Lane 1: molecular mass standards. Lane 2: 5% of theinput (1 μM GST+1000 fold excess E. coli extract). Lane 3: GST retainedby TentaGel-Nbsa-Nlys-Nbsa-Npip-Nlys. Lane 4: GST retained byTentaGel-displayed Nmba-Nbsa-Nleu-Nlys-Npip-Nmba-Nleu-Nleu (the controlpeptoid). Lane 5: GST retained by TentaGel beads without a displayedpeptoid (beads only control). (FIG. 20C) Dilution experiment measuringthe capture of Texas-Red-labeled protein by TentaGel-displayedNbsa-Nlys-Nbsa-Npip-Nlys at the protein concentrations indicated. Allsolutions contained a 100-fold excess of E. coli extract.

FIG. 21 illustrates an exemplary peptide sub-monomer scheme.

FIG. 22 illustrates at the top: a structure of the chalcone-cappedpeptoid library. The linker consisted of a long polyethylene glycolchain to minimize non-specific protein binding. At the bottom: aminesused for the synthesis of the library along with their designations.

FIGS. 23A-23C illustrate a sequence of experiments leading to theidentification of MBP-MDM2 chimeric binding element. (FIG. 23AFluorescence micrograph of the “hit” bead mixed with a population ofsorted library beads; (FIG. 23B) Edman trace of the chalcone-peptoidchimera from the single “hit” bead and (FIG. 23C) its structure aselucidated by Edman sequencing.

FIGS. 24A-24F illustrate the characterization of the binding propertiesof chimeric binding element general formula 2 and related compounds insolution and immobilized on Tentagel beads. Top: Isothermal titrationcalorimetry data for the titration of (FIG. 24A) chalcone-peptoidformula 2 and (FIG. 24B) the peptoidNH₂-Nlys-Npip-Nlys-Nser-Nlys-Nlys-Nlys-Nlys-Npip-Npip lacking thechalcone cap with MBP-Mdm2 and (FIG. 24C) chalcone-peptoid formula 2with MBP alone. The equilibrium dissociation constants derived fromthese data are shown. Bottom (FIGS. 24D-24F): Tentagel beads displayingthe compound indicated were incubated with the Texas Red-labeledproteins indicated and, after washing, the beads were mixed andphotographed under a fluorescence microscope.

FIG. 25 is photomicrographs of Tentagel beads displaying the chimericbinding element of formula 2 after incubation with the indicatedconcentration of Texas Red-labeled MBP-Mdm2 protein followed by thoroughwashing.

FIGS. 26A-26C illustrate the characterization of the solution andsolid-phase binding properties of the ubiquitin lead peptide(NH₂-WGLRALESRWDRYYF) and the chimeric binding element(NH₂-WGLRALESRWDRYYF) and a control peptide. (FIG. 26A) Solutiontitrations of the lead peptide (left) and the chimeric binding element(right) with ubiquitin, monitored by ITC. The equilibrium dissociationconstants calculated from these data are shown. (FIG. 26B) Determinationof the apparent affinity of the indicated immobilized peptides forubiquitin. The beads were incubated with the indicated amount of nativeubiquitin, washed thoroughly, then probed with Texas Red-labeledanti-ubiquitin antibody. After another wash, the beads were photographedunder a fluorescence microscope. (FIG. 26C) Direct comparison of beadsdisplaying the chimeric binding element or no peptide at all (left) andbeads displaying the chimeric binding element and a control peptide(right).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Embodiments of the invention include compositions and methods related tothe develop of high affinity mixed element capture agents (MECAs). Oneaspect of the invention uses a randomized distribution of bindingelements to provide a mixed element capture agent (MECA). Another aspectof the invention uses a low affinity binding element in conjunction witha randomized oligomer of additional binding elements to form a chimericbinding element. A chimeric binding element may then be randomlydistributed on a support to form a MECA.

I. Mixed Element Capture Agents (MECA)

As described above, there is a need for detecting substances from avariety of samples in a rapid high-throughput (HTP) format using methodsand compositions comprising high-affinity and high-specificity captureagents. Most efforts in this area have focused on the use of a singlehigh-affinity biomolecule, such as antibodies, as capture agents.Furthermore, methods for identifying a single high-affinity captureagent are time consuming and laborious. High affinity capture agents ofthe invention described herein include capture agents based on highaffinity interactions produced by concomitant binding of two or more lowto moderate affinity binding elements referred to as a mixed or multipleelement capture agent (MECA). Various embodiments of the inventiondescribed herein provide methods and compositions for production ofsynthetic high-affinity MECA and compositions incorporating MECA(s).There are many reasons to explore synthetic, relatively low molecularmass molecules as capture agents. For example, synthetic molecules areeasier to produce in large quantities with efficient quality control andcan easily be tailored to allow attachment to surfaces in a definedmanner. The invention provides an easy, fast, reproducible and costeffective method of screening, detecting and characterizing variouscomponents from a variety of samples.

It is known that by properly tethering together two modest affinity,non-competitive ligands by use of a linker molecule, a high affinitychimeric ligand can be obtained. However, to achieve this, a linker witha particular length and geometry is required. Current methods toidentify optimal linkers are relatively tedious and labor-intensive,grossly limiting the application of this strategy to high volumeapplications such as the construction of protein-detecting arrays. Asdescribed herein, a novel approach to construct high affinity captureagents without the need for linker synthesis and optimization isdisclosed.

By obviating the need for linker synthesis and optimization, bindingelements with modest binding affinity may be incorporated into highaffinity capture agents (i.e., MECAs). This is accomplished byimmobilizing binding elements on a surface that provides a library ofnumerous geometries between binding elements. For example, if one wereto affix two protein-binding ligands on a surface in a random fashionand at high density, a fraction of the pairs of immobilized moleculeswould be oriented appropriately for binding to a given target.Furthermore, since this technique can use simple synthetic molecules(e.g., peptides and peptide-like compounds) one eliminates the need forthe production and purification of large numbers of biologicalprotein-binding elements, which is far more labor-intensive than smallmolecule synthesis. Although, in certain embodiments, recombinantpeptides or polypeptides may be used as binding elements.

Typically, synthetic molecules do not bind a target with an affinitynecessary for detecting substances in a sample. The composition andmethods described herein may utilize a low to moderate affinitysynthetic molecules to produce MECAs and provide not only timeefficient, but also cost effective high-affinity capture agentcompositions and detection methods. The speed with which new captureagents can be isolated and produced makes the invention an attractivepathway to diagnostic tools, especially when fast diagnosis and highthroughput analysis of a sample(s) is needed.

While applicable to any kind of molecule, a particular embodiment of theinvention uses synthetic molecules as binding elements. Syntheticmolecules may be isolated from combinatorial libraries and generally donot have sufficient affinity as a single binding element to serve asuseful capture agents (for exemplary molecules for use in combinatoriallibraries see Eichler et al., 1995; Cho et al., 1999; LePlae et al.,2002; Ostergaard and Holm, 1997; and Yang et al., 1999 each of which isincorporated herein by reference). Furthermore, synthetic molecules aremuch cheaper and easier to make in quantity than macromolecularprotein-binding compounds such as antibodies. This is one of a varietyof advantages in the production of compositions and devices, asdescribed herein, for the parallel analysis of hundreds of proteinssimultaneously.

In certain embodiments, the invention describes the use of syntheticmolecules as protein MECAs, which was not previously possible due totheir low affinity and the tedious nature of transforming low affinityprotein-binding elements into high affinity ligands. This technologywill typically render small molecules the equal of good antibodies ornucleic acid aptamers in terms of binding affinity. However, smallmolecules are far easier to produce and to adapt as surface-immobilizedprotein ligands than these macromolecular species. This confers numerousadvantages over current macromolecular ligand-based protein detectionmethods, some of which include: (a) high throughput, robust technologythat makes it a frontrunner in the military, medical and research field;(b) suitable for capture of low abundance polypeptides in the sample;(c) small molecule ligands that may be used in this technology typicallyhave the advantage of being more robust than macromolecular proteinligands or nucleic acid ligands (this gives this technology an edge overcurrently available technologies in large-scale production forcommercialization); and (d) in addition, this technology is typicallynot as labor-intensive and is cost effective.

In various embodiments, methods for rapid isolation of MECA(s) forpolypeptide or peptide targets or any other target moleculesparticularly those that can be described as an oligomer of linkedmonomers, such as polysaccharides or nucleic acids are contemplated. Thecompositions and methods described herein may be used to detect apolypeptide in a sample, via the high affinity binding of thepolypeptide to two or more surface-immobilized binding elements (MECA).A binding element may be isolated from phage, peptide, and/or chemicallibraries and the like.

In examples provided herein, a model system is described in whichimmobilized peptides are used as binding elements to form a modelcapture agent for homodimeric proteins. In this simplest case, there isonly one type of binding element, thus technically not a mixed elementcapture agent, on the surface, but two identical molecules mustcollaborate to bind the target dimer tightly. Simple, linear peptidesthat exhibit modest affinities for their target proteins in solution(K_(D)s in the μM range) act as sub-nanomolar capture agents whenimmobilized on a surface.

In the case where the target protein is a monomer, two different,non-competitive surface-linked binding elements, i.e., a MECA, mustcollaborate to provide high affinity binding. In the case shown in FIGS.9A-9B, this was achieved by co-immobilizing peptides that bind monomericMBP and monomeric mdm2 (in the form of a single fused linear peptide, achimeric binding element), thus providing a high affinity MECA for theefficient capture of the MBP-mdm2 model protein. The fused linearpeptide is referred to as a chimeric binding element because twodistinct binding elements are operatively coupled to each other. Theimmobilized peptide-protein complexes are shown to be long-lived, withlifetimes well in excess of what would be required for aprotein-detecting application. MECAs comprised of two or more bindingelements can bind any protein with high affinity, regardless of itsmolecularity, and is thus a completely general approach to thedevelopment of high affinity capture agents.

In various embodiments of the invention, heteromultimer or heteromericcomplexes may also be bound by MECAs. Each binding element may bind thesame or different component of a heteromultimer. A “heteromultimer” isdefined as a higher order complex of different or heterologous moleculesor substances. For instance, a heteromultimer refers to a complex ofheterologous proteins. A heteromultimer may be an association of 2, 3,4, 5, 6, 7, 8, 9, 10, or more molecules or compounds, e.g., proteins. Insome instances, a component of a heteromultimer may be present one ormore times within the heteromultimer. For example, A is a first proteinand B is a second protein. A heteromultimer may be protein complex witha structure of (BAAB).

Various embodiments of the invention include two or more protein bindingelements that bind with low to modest affinity in solution and act astenacious capture agents when immobilized on a support. Linear peptidesimmobilized on beads are able to capture dimeric proteins from dilute(<1 nM protein) solutions essentially quantitatively (see FIGS. 7A-7B).Furthermore, immobilized peptide/protein complexes have beendemonstrated to have kinetic half-lives of several hours. Thespecificity of MECA binding rivals that of a good antibody. Simplemolecules isolated directly from various readily available combinatoriallibraries can be used as practical capture agents for a capture target,for example, a monomeric, homodimeric or higher-order protein(s).

In certain embodiments, two low to moderate affinity, non-competingbinding elements may be co-immobilized on a surface. For example, acombination of binding elements for a variety of molecules with almostany desired distance between them may be identified on a surface.Consequently, two or more binding elements will be appropriately spacedto form high-affinity binding site(s) on some fraction of the surface.One can envision that the higher the immobilized binding elementdensity, the greater the fraction of the surface would representhigh-affinity binding sites.

Examples of how this may be implemented to provide high affinityimmobilized capture agents include two binding elements that aredifferent and non-competitive bind to different surfaces of a monomericprotein. The different surfaces could be different domains of amulti-domain protein or different surface features on a single domain.Note that one could distinguish between splice variants of a proteinusing this approach (see FIG. 2 for an example). Another aspect includestwo binding elements bind different proteins which are part of the samemultiprotein complex (see FIGS. 3A-3D for an example). Still anotheraspect includes one binding element that recognizes the polypeptide,while a second binding element recognizes a particularpost-translational modification (phosphorylation, ubiquitination,glycoslyation, etc.) (see FIG. 4 for an example). Thus the two bindingelements would cooperate to bind tightly to a modified form of a proteinand not to an unmodified protein.

Surface-immobilized capture agents may be one-half or a portion of adevice for the sensitive detection of a target, e.g., proteins. Theother half or portions would comprise a method to quantify the amount oftarget(s) captured by the capture agent. There are many ways this couldbe accomplished, for example using a “sandwich assay” in which thecaptured proteins are probed with a fluorescently labeled antibodyagainst the protein of interest. The level of bound fluorescence, whichcorresponds to the level of the captured protein, would then bemonitored using a number of commercially available instruments. Luminex(Austin, Tex.) as well as other companies market instruments that enablethis type of assay to be done in a high-throughput format. Anotherpossibility is to immobilize the binding elements on a surface capableof intrinsically sensing the binding of the target molecule. Forexample, a modified gold surface, which would allow surface plasmonresonance (SPR) to be used to quantify binding. Many other detectionschemes, some of which are described below, are possible and it isimportant to note that the invention, which focuses on the developmentof novel capture agents, could be wedded to any of them.

Certain embodiments of the invention relate to compositions and methodsfor detection of a target or capture target. A detector may comprise oneor more sensing elements or regions of a support. In some embodiments,for example when the support is a planar surface, a support may includesegregated areas separating sensing elements. The segregated sensingelements or regions may include one or more MECAs. In some embodiments,one or more sensing elements or regions for a number of targets arecontemplated. A support may contain one, tens, hundreds, or thousands ofsensing elements. One or more supports may be incorporated in or used inconjunction with a detection means to form a detector. These detectorsmay be useful in methods of analyzing complex mixtures of substancessuch as clinical samples or cell extracts, as well as gaseous orvolatile substances of both biological and non-biological origin.

A detector may comprise a support surface on which capture agents areimmobilized (e.g., peptides or proteins). The building blocks of adetector and related methods include surface chemistry (the chemicalbonds that immobilize binding elements), sample(s) (substances used tostudy interactions, compositions or expression levels), capture agents(agents used to capture and quantify target(s)) and detection methods(methods used to detect binding to or interact with capture agents onthe surface of support).

Embodiments of the invention may have several uses in research,medicine, military, forensics and other fields. In general, almost anyapplication (e.g., diagnostics) that currently uses immobilizedantibodies could employ these capture agents, which are superior toantibodies in many ways. Furthermore, proteomics devices, such asprotein-detecting microarrays designed to monitor the levels of hundredsof proteins simultaneously, would be greatly enabled by the existence ofthese capture agents. Currently, most such devices are based uponbiological macromolecules such as antibodies or nucleic acid aptamers,which are difficult to produce in large quantities and can often loseactivity when attached to a surface. A variety of the capture agentsdescribed herein may be produced in bulk and at less cost, as well asbeing stable in a variety of conditions and for extended periods oftime.

For example, in medicine, MECAs may detect the presence or absence ofmultiple biological markers in complex samples such as blood or urine,providing a valuable diagnostic tool. In the military, the presence ofcertain polypeptides can signal the presence of a biological warfareagent in the environment. In the research field, it is an efficient andfast screening/detection tool. The invention as described herein doesnot require any prior knowledge of the polypeptide, molecule, orsubstance to be bound to the MECAs. Binding elements may be identifiedby screening various combinatorial or expression libraries or othertypes of compound collections.

Compositions and methods disclosed herein may include “chimeric bindingelements,” in particular chimeric binding elements that include a firstknown low to moderate affinity binding element and a second oligomericcomponent that provides a second binding element. Typically, a chimericbinding element is identified by screening a plurality of oligomericcompounds coupled to the known binding element. Certain chimeric bindingelements provide for the rapid transformation of low to moderateaffinity binding elements into high affinity capture agents. Embodimentsof the invention also include a facile method to obtain high affinitysynthetic protein capture agents. Various embodiments of the chimericbinding element approach is a simple and efficient route to suchmolecules is described herein.

Relatively low molecular weight synthetic species may be isolated thatare shown to capture a target protein, e.g., Mdm2 and/or Ubiquitin, fromsolutions containing a large excess of other proteins. A semi-automatedscreening protocol is described below that makes this system capable ofhigh throughput. The invention provides for screening combinatorialchemical libraries or large compound collections for chimeric bindingelements with a reasonable assurance of success.

Most ligands obtained from screening naïve libraries bind their targetprotein with equilibrium dissociation constants (K_(D)s) in the μMrange. Such modest affinity ligands are suitable for some applicationssuch as chemical genetics studies (Schreiber, 2003; Koehler et al.,2003; Kuruvilla et al., 2002), but for other important applications muchhigher affinity is required. For example, there is considerable interestin the development of protein-detecting microarrays based on smallmolecule capture agents attached to a modified glass slide, encodedbeads or some other suitable surface (for reviews, see Kodadek, 2001;2002). In order for such devices to be applied to the analysis of lowabundance proteins, the synthetic capture agents must exhibit bindingproperties comparable to that of a good monoclonal antibody, formingcomplexes with K_(D)s in the 10⁻⁸ M to 10⁻¹² M range. To evolve a modestaffinity lead compound to a high affinity capture agent by traditionalmedicinal chemistry methods is time-consuming and labor-intensive, whichmake it difficult to apply on a proteomic scale.

The inventors describe a rapid and economical method to transform modestaffinity lead compounds into high affinity agents. Typically, theprotocol involves capping a combinatorial library of oligomericcompounds, such as peptides, peptoids or other types of molecules thatmay be used to produce combinatorial libraries, with the lead compound(i.e., first binding element) and screening this library against thetarget protein under conditions too demanding for the first bindingelement to support binding. The screen will simultaneously select for asecond binding element within the oligomer and a suitable linkerconnecting this element and the lead compound, thus providing a MECAcomprising a chimeric binding element. The efficacy of this approach maybe demonstrated with the isolation and characterization of two syntheticmolecules that are able to capture Mdm2 protein and ubiquitin,respectively, from complex solutions containing a large excess of otherproteins even when the target is present at nanomolar concentrations,see Examples below.

II. Supports for Immobilizing Binding Elements

In various embodiments of the invention binding elements and MECAs maybe operatively coupled to a support. “Support” refers to a solid phaseonto which a MECA can be provided, (e.g., by attachment, deposition,coupling and other known methods). One or more binding elements may beimmobilized on supports including, but not limited to glass (e.g., achemically modified glass slide), latex, plastic, membranes, microtiter,wells, mass spectrometer plates, beads (e.g., cross-linked polymerbeads) or the like.

In one aspect, the invention provides supports adapted for use with adetector or a detection method(s) (e.g., ELISA), wherein the supportcomprises a MECA immobilized on the support surface. The MECA(s) willtypically bind with a high affinity and specificity to a component of asample. In various non-limiting embodiments the sample is a biologicalsample. The component may be involved in a biological pathway (e.g.,signal transduction, immunological response, cytoplasmic or membraneenzyme mediated pathway, cell cycle or developmental cycle pathway).Typically, MECAs are located at different addressable, segregatedregions referred to as sensing elements or regions on a support so thatone can readily distinguish which components in a sample are bound to asupport. In some embodiments, MECAs can be placed in the same sensingelement or region of the support as long as the components can bedifferentially detected. The supports and the MECAs are described indetail herein.

Targets or capture targets can be captured on any of a variety ofMECA/support. One of the various MECA/support is a protein biochip.Among the many protein biochips described in the art are those biochipsproduced by Ciphergen Biosystems (Fremont, Calif.), Packard BioScienceCompany (Meriden Conn.), Zyomyx (Hayward, Calif.) and Phylos (Lexington,Mass.). In general, protein biochips comprise a support having agenerally planar surface. A binding element(s) and/or a MECA(s) istypically attached to the surface of the support. In certain embodimentsof the invention a surface may comprise a plurality of addressablelocations, each of which location has one or more binding elements boundto form a MECA. The binding element can be a biological molecule, suchas a peptide, polypeptide or a nucleic acid, which binds otherbiomolecules in a specific manner. Examples of protein biochips aredescribed in the following patents or patent applications: U.S. Pat.Nos. 6,225,047 and 6,329,209, and International publication WO 99/51773and WO 00/56934, each of which is incorporated herein by reference.

In one embodiment the support is capable of being engaged by aninterface of a mass spectrometer which positions the support in aninterrogatable relationship with an ionization source. The support canbe in any shape, e.g., in the form of a strip, a plate, or a dish with aseries of wells. Each MECA(s) may be immobilized at differentaddressable locations at the support surface.

Typically, each sensing element or region comprises a different MECA sothat one can readily distinguish which target(s) in a sample is/arebound to the support. In some embodiments, different MECAs can beproduced in the same sensing region of the support as long as the MECAshave different detectable characteristics.

Each sensing region on the support may be “addressable” in that duringdetection of capture target binding, a detection method may be directedto, or “addresses” the sensing region where a capture target is bound tothe MECA. The addressable locations can be arranged in any pattern onthe support, but are preferably in regular pattern, such as lines,orthogonal arrays, or regular curves (e.g., circles). Alternatively,MECAs can be placed on the support surface in continuous patterns,rather than in discontinuous patterns.

Alternatively, the support can be a separate material. For example, asupport can be a solid phase, such as a polymeric, paramagnetic, latexor glass bead, upon which are immobilized binding elements, whichproduce a MECA for binding a target. A solid phase material may beplaced onto a probe or detectable media (e.g., fluorescently taggedbead) that is removably insertable into a gas phase ion spectrometer orpassed by a detector such as a laser/spectrometer device. The solidphase with each type of capture agent is typically placed at differentaddressable locations of the support surface. Alternatively, as notedabove, different capture agents can be placed on the same addressablelocations as long as they are able to be differentially detected.

The support can be also shaped so that it is adapted for use withvarious components of a gas phase ions spectrometer, such as inletsystems and detectors. For example, the support can be adapted formounting in a horizontally and/or vertically translatable carriage thathorizontally and/or vertically moves the support to a successiveposition. This allows components bound to different locations of thesupport surface to be analyzed without requiring repositioning of thesupport by hand.

The support can be made of any suitable material. For example, thesupport materials include, but are not limited to, insulating materials(e.g., glass such as silicon oxide, plastic, ceramic), semi-conductingmaterials (e.g. silicon wafers), or electrically conducting materials(e.g., metals, such as nickel, brass, steel, aluminum, gold, orelectrically conductive polymers), organic polymers, biopolymers, or anycombination thereof. The support material can also be solid or porous.Examples of supports suitable for use in embodiments of the inventionare described in U.S. Pat. No. 5,617,060 and International PublicationWO 98/59360, each of which are incorporated by reference.

The support can be conditioned to bind binding elements. In someembodiments, the surface of the support can be conditioned (e.g.,chemically or mechanically such as roughening) to place binding elementson the surface. Typically, a support comprises reactive groups that canimmobilize binding elements. For example, the support can comprise acarbonyldiimidazole group which covalently reacts with amine groups ofnucleic acids or peptides. In another example, the support can comprisean epoxy surface which covalently reacts with amine and thiol groups ofDNA and proteins. Supports with these reactive surfaces are commerciallyavailable from Ciphergen Biosystems (Fremont, Calif.).

III. Binding Elements

Binding elements are molecules or portions of molecules that demonstratean affinity for a particular target, which may or may not bind with ahigh enough affinity to capture a particular target. Binding elementsare typically operatively coupled to a support as described herein andmay be part of a MECA. In other embodiments, a binding element includesa chimeric binding element wherein two or more binding elements areoperatively coupled. Typically, high affinity binding of a target willresult from concomitant binding of two or more chimeric binding elementsto a single target. As used herein, these binding elements include lowand/or moderate affinity binding elements. “Low affinity,” as usedherein, is defined as an interaction with a dissociation constant(K_(D)) of ≧10⁻⁵ M, “moderate affinity” as used herein is defined as aK_(D) between 10⁻⁵ M and 10⁻⁸ M, and “high affinity” as used herein isdefined as a K_(D) of M. Binding elements used to form a MECA may be anymolecule or substance that binds a target molecule (capture target) withpreferably a low to moderate affinity. In various embodiments a bindingelement(s) may include, but is not limited to a peptide, a peptoid(i.e., N-substituted oligoglycines), a peptide-like molecule, apolypeptide, a protein, a polysaccharide, a nucleic acid, a smallmolecule, an inorganic molecule, an organic molecule, a single chainantibody or the like. It is also contemplated that combinations ofdifferent classes of binding elements may also be used, for example, apeptide modified with a small molecule and the like. Capping moleculesused to form chimeric binding elements may be any molecule or substancethat binds a target molecule with a low to moderate affinity. In variousembodiments a capping molecule may include, but is not limited to apeptide, a peptoid (i.e., N-substituted oligoglycines), a peptide-likemolecule, a polypeptide, a protein, a polysaccharide, a nucleic acid, asmall molecule, an inorganic molecule, an organic molecule, a singlechain antibody or the like. It is contemplated that combinations ofdifferent classes of binding elements may be used in forming a chimericbinding element, for example, a peptoid with a small molecule as acapping molecule and the like. Binding elements may be identified by avariety of known methods including, but not limited to combinatorial,expression, phage display, yeast two hybrid libraries and the like. Oneor more binding element may be used to form a MECA as described herein.In some embodiments, binding elements may be covalently coupled or fusedto each other, for example a fusion of two peptides, with or withoutintervening residues, into a single linear molecule, i.e., a chimericbinding element. For each MECA, a preferred density may be empiricallydetermined by arraying a number of sensing elements, which include oneor more binding elements, at varying densities and identifying anoptimal binding element density. In some embodiments, the individualbinding elements could be anchored in a fluid membrane, such a lipidbilayer, allowing them to reorient so as to achieve proper spacing toact as a high affinity MECA.

In certain embodiments, a binding element may be derived from abiological pathway(s) of interest. Biological pathways include, but arenot limited to components such as metabolites, intermediates,polypeptides, lipids, lipoproteins, carbohydrates, and nucleic acids.Binding elements may be selected from any suitable materials as long asthey bind to a target. For example, binding elements may be selectedfrom polypeptides, lipids, lipoproteins, carbohydrates, nucleic acids,steroids, glycolipids, small organic, inorganic molecules and/orderivatives thereof. Typically, binding elements may include, but arenot limited to entities that bind proteins, receptors, ligands,antibodies, organelles, microbes, pathogens, and nucleic acids.

One or more different types of binding elements and/or MECA may beimmobilized on a support surface. MECAs may be localized or segregatedto particular regions on a support or on particular supports, e.g.,latex beads. Each of these particular regions will be able to bind atleast one target. These regions are referred to as sensing elements orregions. Typically, at least 2, 3, 4, 5, 6, 7, 8 or more differentbinding elements, or even hundreds or thousands of different bindingelements can be immobilized on a support surface to form various MECAs.MECAs can be selected to bind to a single target or multiple targets.

In certain embodiments, a target(s) may be indicative of a particularstate of a non-biological or biological substance, sample or pathway.For example, many signal transduction pathways and their components areknown, and the selection of a MECA depends on analysis of which signaltransduction pathway(s) are to be monitored. For example, bindingelements and/or MECAs may be selected from those that selectively bindto components of the Ras/Raf signal transduction pathway, the p53 tumorsuppressor signal transduction pathway, the BRCA1 signal transductionpathway, a pathogen or pathogen bi-product, a marker related to adisease state or any combination thereof. Many other signal transductionpathways are known in the art, and are described in, e.g., Alberts etal. (1994) and Lodish et al. (2000). In other embodiments, a MECA may bechosen to detect the presence or absence of one or more pathogen, suchas bacteria, viruses, parasites or a portion or by product thereof.

In certain aspects, MECAs may be selected so that a number of MECAs bindto a target or to components of a target pathway, disease or organism.An array of MECAs may be selected so that at least two different MECAson the support surface bind to components that are sequential in theiractivation in a signal transduction or other biologic/non-biologicpathway. Having a number of MECAs that bind to components of a singlepathway, disease or organism on a support allows those skilled in theart to readily determine which component in a sample is, for exampledefective. In some embodiments, a sample may be related tonormal/non-normal cell development, normal/disease condition,infected/non-infected condition, presence/absence of an organism/agentand the like.

A capping molecule or first binding element of a chimeric bindingelement may be coupled to a monomer, polymer or oligomer of at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore units that will vary in order and compositions from candidateelement to candidate element. Each unit may be an amino acid, a nucleicacid, a saccharide, an inorganic molecule, organic molecule, or acombination thereof, including various derivatives thereof. Thus,providing a library of candidates from which a chimeric binding elementmay be selected.

A. Peptides and Peptide Like Molecules

In various aspects of the invention, peptides, peptoids, polypeptides,and/or proteins may be used as a binding element or as a portion of achimeric binding element. The peptides, polypeptides and/or proteinsused as a binding element or an oligomer in a chimeric binding elementmay be an isolated, a recombinant or a synthetic peptide(s), peptoid(s),polypeptide(s), proteins and/or other oligomeric molecule. A peptide,peptoid or polypeptide may be identified as a binding element by variousknown methods, such as phage display library, combinatorial peptide orpeptoid library, classical binding studies and the like. Typically, thecomposition of a peptide, peptoid, polypeptide or other oligomer will bevariable and, in certain embodiments, will be operatively coupled with acapping molecule and/or first binding element to form a library ofcandidate chimeric binding elements.

The present invention may also relate to fragments of a polypeptide.Fragments, including the N-terminus of the molecule may be generated bygenetic engineering of translation stop sites within a coding region.Alternatively, treatment of polypeptide with proteolytic enzymes, knownas proteases, can produce a variety of N-terminal, C-terminal andinternal fragments. In certain embodiments, peptides or peptide likemolecule, i.e., peptoids, may be synthesized by known methods. Examplesof fragments may include contiguous residues of 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, 100, 200, 300 or more aminoacids, amino acid mimetics, derivatives, or combinations thereof. Thesefragments may be purified according to known methods, such asprecipitation (e.g., ammonium sulfate), HPLC, ion exchangechromatography, affinity chromatography (including immunoaffinitychromatography) or various size separations (sedimentation, gelelectrophoresis, gel filtration).

B. Synthetic Peptides

Various embodiments of the invention describe peptides or peptidemimetics for use in the production of MECAs. Peptides, peptide mimeticsor peptide like molecules of the invention may also be synthesized insolution or on a solid support in accordance with conventionaltechniques. Various automatic synthesizers are commercially availableand can be used in accordance with known protocols. See, for example,Stewart and Young (1984); Tam et al. (1983); Merrifield (1986); andBarany and Merrifield (1979), each incorporated herein by reference.Short peptide or oligomeric sequences, or libraries of overlappingpeptides or oligomers, usually from about 6 up to about 35 to 50 aminoacids or monomers, which correspond to binding elements or secondbinding elements of chimeric binding elements described herein, can bereadily synthesized and then screened in screening assays designed toidentify molecules, peptides, peptide mimetics, or other oligomer ofinterest. In some embodiments, recombinant DNA technology may beemployed wherein a nucleotide sequence which encodes a peptide of theinvention is inserted into an expression vector, transformed ortransfected into an appropriate host cell and cultivated underconditions suitable for expression.

C. Fusion Peptides

A specialized kind of insertional variant is the fusion protein orpeptide. This molecule generally has all, a substantial portion, or aportion of a first molecule, linked at the N- or C-terminus, to all or aportion of a second molecule. For example, fusions typically employleader sequences from other species to permit the recombinant expressionof a protein in a heterologous host. Other useful fusions includelinking of binding elements. Fusions of the invention include a fusionof two or more binding elements. In certain embodiments the two or moreelements are reversibly or irreversibly coupled to each other.

D. Purification of Peptides

In certain embodiments, it may be desirable to purify a peptide orpolypeptide. Protein and peptide purification techniques are well knownto those of skill in the art. These techniques involve, at one level,the crude fractionation of the cellular milieu to polypeptide andnon-polypeptide fractions. Having separated the polypeptide from otherproteins, the polypeptide of interest may be further purified usingchromatographic and electrophoretic techniques to achieve partial orcomplete purification (or purification to homogeneity). Analyticalmethods particularly suited to the preparation of a pure peptide orpeptide like molecule are ion-exchange chromatography, exclusionchromatography; polyacrylamide gel electrophoresis; isoelectricfocusing. A particularly efficient method of purifying peptides orpeptide like molecules is fast protein liquid chromatography or evenHPLC. These purification techniques may be used to purify other bindingelements or portions of chimeric binding elements.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of a peptide,peptide like molecule, or other binding element. The term “purifiedpeptide, peptide like molecule, polypeptide or binding element” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the peptide is purified to any degree relative toits naturally-obtainable state. A purified protein, peptide, or bindingelement therefore also refers to a protein, peptide, or binding element,free from the environment in which it may naturally occur.

Generally, “purified” will refer to a protein, peptide, or bindingelement composition that has been subjected to fractionation to removevarious other components, and which composition substantially retainsits activity. Where the term “substantially purified” is used, thisdesignation will refer to a composition in which the protein, peptide,or binding element forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the peptides, peptide like molecules, polypeptides,or binding elements in the composition. Various methods for quantifyingthe degree of purification will be known to those of skill in the art inlight of the present disclosure.

Various techniques suitable for use in purification will be well knownto those of skill in the art. These include, for example, precipitationwith ammonium sulphate, PEG, antibodies and the like or by heatdenaturation, followed by centrifugation; chromatography steps such asion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified peptide or binding element.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (alter pH, ionic strength, temperature, etc.).

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand should alsoprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

E. Monoclonal and Single Chain Antibodies

Binding elements may be antibodies that bind to a target. A majority ofsingle chain antibodies do not bind a target with an affinity sufficientfor the antibody to be used as a capture agent. Therefore, certainembodiments of the invention may utilize sub-optimal single chainantibodies to produce a capture agent. In various aspects of theinvention antibodies that demonstrate a low or moderate affinity for atarget may be used as a binding element to produce a capture agent.These include, e.g., monoclonal antibodies, antibody fragments, singlechain antibodies, and the like. Methods for making these molecules arewell-known in the art.

For example, monoclonal antibodies can be prepared by any technique thatprovides for the production of antibody molecules by continuous celllines in culture, including the hybridoma technique (Kohler andMilstein, 1975), as well as the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983), and the EBV-hybridomatechnique to produce human monoclonal antibodies (Cole et al., 1985).

Fragments of antibodies may also be useful as binding elements. Whilevarious antibody fragments can be obtained by the digestion of an intactantibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by utilizing recombinant DNAmethodology. Thus, the term “antibody,” as used herein, also includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv). Single chain antibodies may alsobe useful in the production of capture agents. Methods for producingsingle chain antibodies were described in, for example, U.S. Pat. No.4,946,778. Techniques for the construction of Fab expression librarieswere described by Huse et al. (1989); these techniques facilitate rapididentification of monoclonal Fab fragments with the desired specificityfor pathway components.

F. Aptamers

In certain embodiments, binding elements may comprise nucleic acids. Asdiscussed below, a nucleic acid may contain a variety of different basesand yet still produce a binding element that may be used in theproduction of a MECA.

The methods of the present invention may select and use nucleic acidsthat bind to a variety of substances with a low to moderate affinity. Incertain embodiments, a nucleic acid may comprise or encode an aptamer.An “aptamer” as used herein refers to a nucleic acid that binds a targetmolecule through interactions or conformations other than those ofnucleic acid annealing/hybridization. Methods for making and modifyingaptamers, and assaying the binding of an aptamer to a target moleculemay be assayed or screened for by any mechanism known to those of skillin the art (see for example, U.S. Pat. Nos. 5,840,867, 5,792,613,5,780,610, 5,756,291 and 5,582,981, incorporated herein by reference).

G. Other Binding Elements

Virtually any molecule or compound having a low to moderate affinity fora target molecule may be used as a binding element and/or a cappingmolecule. Binding elements may include non-biological or biologicalpolymers, polysaccharides, a variety of small molecules, lipids, and thelike.

Methods have been developed for the combinatorial (e.g., rapid-serial orparallel) synthesis and screening of libraries of small molecules ofpharmaceutical interest, and of biological polymers such as peptoids,polypeptides, proteins, oligonucleotides and deoxyribonucleic acid (DNA)polymers (Eichler et al., 1995; Cho et al., 1999; LePlae et al., 2002;Ostergaard and Holm, 1997; Yang et al. 1999). U.S. Pat. Nos. 6,475,391and 6,461,515; and Brocchini et al. describe exemplary methods andcompositions for the preparation and characterization of polymercombinatorial libraries for selecting polymer materials (Brocchini etal., 1997). Exemplary synthetic methods for oligosaccharides is providedin Kanemitsu and Kanie (2002).

Various small molecule libraries may be obtained from commercial ornon-commercial sources, as well as synthesizing such compounds usingstandard chemical synthesis technology or combinatorial synthesistechnology (see U.S. Pat. No. 6,344,334; Gallop et al. (1994), Gordon etal. (1994), Thompson and Ellman (1996), each of which is incorporatedherein by reference).

IV. Methods of Detection

Methods detecting targets captured on a solid support can generally bedivided into photometric methods of detection and non-photometricmethods of detection.

Photometric methods of detection include, without limitation, thosemethods that detect or measure absorbance, fluorescence, refractiveindex, polarization or light scattering. Methods involving absorbanceinclude measuring light absorbance of an analyte directly (increasedabsorbance compared to background) or indirectly (measuring decreasedabsorbance compared to background). Measurement of ultraviolet, visibleand infrared light all are known. Methods involving fluorescence alsoinclude direct and indirect fluorescent measurement. Methods involvingfluorescence include, for example, fluorescent tagging in immunologicalmethods such as ELISA or sandwich assay. Methods involving measuringrefractive index include, for example, surface plasmon resonance(“SPR”), grating coupled methods (e.g., sensors uniform gratingcouplers, wavelength-interrogated optical sensors (“WIOS”) and chirpedgrating couplers), resonant mirror and interferometric techniques.Methods involving measuring polarization include, for example,ellipsometry. Light scattering methods (nephelometry) may also be used.

Non-photometric methods of detection include, without limitation,magnetic resonance imaging, gas phase ion spectrometry, atomic forcemicroscopy and multipolar coupled resonance spectroscopy. Magneticresonance imaging (MRI) is based on the principles of nuclear magneticresonance (NMR), a spectroscopic technique used by scientists to obtainmicroscopic chemical and physical information about molecules, for areview see Hornak, 2002. Gas phase ion spectrometers include massspectrometers, ion mobility spectrometers and total ion currentmeasuring devices.

Mass spectrometers measure a parameter which can be translated intomass-to-charge ratios of ions. Generally ions of interest bear a singlecharge, and mass-to-charge ratios are often simply referred to as mass.Mass spectrometers include an inlet system, an ionization source, an ionoptic assembly, a mass analyzer, and a detector. Several differentionization sources have been used for desorbing and ionizing analytesfrom the surface of a support or biochip in a mass spectrometer. Suchmethodologies include laser desorption/ionization (MALDI, SELDI), fastatom bombardment, plasma desorption and secondary ion massspectrometers. In such mass spectrometers the inlet system comprises asupport interface capable of engaging the support and positioning it ininterrogatable relationship with the ionization source and concurrentlyin communication with the mass spectrometer, e.g., the ion opticassembly, the mass analyzer and the detector.

Solid supports for use in bioassays that have a generally planar surfacefor the capture of targets and adapted for facile use as supports withdetection instruments are generally referred to as biochips. Proteinbiochips are biochips adapted for use in the detection of peptides orproteins or targets captured by proteins.

In certain embodiments, methods for detecting components of a biologicalpathway, e.g., a signal transduction pathway, wherein the methods maycomprise: providing a support comprising at least two different captureagents immobilized on a surface of the support, wherein the captureagents specifically bind to a target component(s) of a biologicalpathway, contacting a sample with a support, and detecting thecomponents of the biological pathway bound to their correspondingcapture agents on the support by gas phase ion spectrometry. In someembodiments, data generated by gas phase ion spectrometry from a testsample can be compared to a control to determine if there is any defectin the biological pathway in the test sample. The sample preparationmethods and gas phase ion spectrometry analysis are described in U.S.Patent Application 20020137106, incorporated herein by reference.

V. Sample Preparation and Handling

The sample used in this invention can be derived from essentially anysource. In particular embodiments the sample may be derived from abiological source. These include, e.g., body fluids such as blood,feces, sputum, urine, serum, saliva, or extracts from biologicalsamples, such as bacterial or cell lysates. In certain embodiments, asample is in liquid form. In some embodiments a sample may be derivedfrom a gas sample, such as an air sample.

The sample is contacted with a support comprising a capture agent in anysuitable manner, e.g., bathing, soaking, dipping, spraying, washingover, or pipetting. Generally, a volume of sample containing from 1 pMto 1 mM of a capture target in a volume from about 1 μl to 1 ml issufficient for binding to the capture agent. The sample can contact thesupport comprising one or more capture agents for a period of timesufficient to allow the target molecules to bind to the capture agent.Typically, the sample and the support comprising the capture agents arecontacted for a period of between about 30 seconds and about 12 hours.In some embodiments, between about 30 seconds and about 15 minutes.Typically, the sample is contacted with the capture agent under ambienttemperature and pressure conditions. For some samples, however, modifiedtemperature (typically 4° C. through 37° C.) and pressure conditions maybe desirable. These conditions are determinable by those skilled in theart.

After the support contacts the sample or sample solution, it ispreferred that unbound and weakly absorbed materials on the supportsurface are washed out or off so that only the tightly bound materialsremain on the support surface. Washing a support surface can beaccomplished by, e.g., bathing, soaking, dipping, rinsing, spraying, orwashing the support surface with an eluant. A microfluidics process maybe used when an eluant is introduced to small spots of capture agents onthe support. Typically, an eluant may be at a temperature of between 0°C. and 100° C. or between 4° C. and 37° C. In some embodiments, washingunbound materials from the probe surface may not be necessary if pathwaycomponents bound on the probe surface can be resolved by gas phase ionspectrometry without a wash or are detected using a high specificitysandwich reagent that will ignore molecules that might be present otherthan the target.

Any suitable eluants (e.g., organic or aqueous) that preserve therelevant interaction can be used to wash the support surface.Preferably, an aqueous solution is used. Exemplary aqueous solutionsinclude, e.g., a HEPES buffer, a Tris buffer, or a phosphate bufferedsaline. To increase the wash stringency of the buffers, additives can beincorporated into the buffers. These include, but are limited to, ionicinteraction modifier (both ionic strength and pH), hydrophobicinteraction modifier, chaotropic reagents, affinity interactiondisplacers. Specific examples of these additives can be found in, e.g.,PCT publication WO98/59360. The selection of a particular eluant oreluant additives is dependent on experimental conditions (e.g., types ofcapture agents used or biological pathway, e.g., signal transduction,immunological, plasma membrane enzyme mediated, cell cycle ordevelopmental cycle components to be detected), and can be determined bythose of skill in the art.

Prior to desorption and ionization of a target from a support surface,an energy absorbing molecule (“EAM”) or a matrix material is typicallyapplied to the support surface. The energy absorbing molecules canassist absorption of energy from an energy source from a gas phase ionspectrometer, and can assist desorption of targets from the supportsurface. Exemplary energy absorbing molecules include cinnamic acidderivatives, sinapinic acid (“SPA”), cyano hydroxy cinnamic acid(“CHCA”) and dihydroxybenzoic acid. Other suitable energy absorbingmolecules are known to those skilled in the art. See, e.g., U.S. Pat.No. 5,719,060 for additional description of energy absorbing molecules.

The energy absorbing molecule and the sample can be contacted in anysuitable manner. For example, an energy absorbing molecule is mixed withthe sample, and the mixture is placed on the support surface. In anotherexample, an energy absorbing molecule can be placed on the supportsurface prior to contacting the support surface with the sample. Inanother example, the sample can be placed on the support surface priorto contacting the support surface with an energy absorbing molecule.Then the components bound to the capture reagents on the support surfaceare desorbed, ionized and detected as described in detail below.

VI. Analysis of Data

Data generated by desorption and detection of a bound target (e.g.,signal transduction, immunological, plasma membrane enzyme mediated,cell cycle, developmental cycle, pathogen components) can be analyzedusing any suitable means. In one embodiment, data is analyzed with theuse of a programmable digital computer. The computer program generallycontains a readable medium that stores codes. Certain code can bedevoted to memory that includes the location of each feature on asupport, the identity of the capture agents at that feature and theelution conditions used to wash the support surface. The computer alsocontains code that receives as input, data on the strength of the signalat various addressable locations on the support. This data can indicatethe number of targets detected, including the strength of the signalgenerated by each target.

Data analysis can include the steps of determining signal strength(e.g., height of peaks) of a target(s) detected and removing “outliers”(data deviating from a predetermined statistical distribution). Theobserved peaks can be normalized, a process whereby the height of eachpeak relative to some reference is calculated. For example, a referencecan be background noise generated by instrument and chemicals (e.g.,energy absorbing molecule) which is set as zero in the scale. Then thesignal strength detected for each target can be displayed in the form ofrelative intensities in the scale desired. Alternatively, a standard maybe admitted with the sample so that a peak from the standard can be usedas a reference to calculate relative intensities of the signals observedfor each target detected.

Data generated by desorption and detection of target(s) in a test samplecan be compared to control data to determine if the target(s) in thetest sample is normal. A control data refers to data obtained fromcomparable samples from a normal cell, sample or person, which or who isknown to have defined profile with regard to target molecules or sampleconditions. For each target being analyzed, a control amount of eachcomponent from a normal or standardized sample is determined.Preferably, the control amount of each target is determined based upon asignificant number of samples taken from samples such as normal cells orpersons so that it reflects variations of the amount of these targetsseen in the normal cell or population.

If the test amount of a particular target is significantly increased ordecreased compared to the control amount of the component, then this isa positive indication that the test sample has an underlying defect orcontains a particular test substance or organism. For example, if thetest amount of a biological pathway component is increased or decreasedby at least 1.5-fold, 2-fold, 5-fold or 10-fold compared to the controlamount, then this is an indication that the test sample has a defect inthe biological pathway.

Data generated by the detector, e.g., the mass spectrometer, can then beanalyzed by computer software. The software can comprise code thatconverts signal from the detector into computer readable form. Thesoftware also can include code that applies an algorithm to the analysisof the signal to determine whether the signal represents a “peak” in thesignal corresponding to a target. The software also can include codethat executes an algorithm that compares signal from a test sample to atypical signal characteristic of “normal” and determines the closenessof fit between the two signals. The software also can include codeindicating whether the test sample has a normal profile of the target(s)or if it has an abnormal profile.

VII. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Mixed Element Capture Element Studies Materials and Methods

Plasmids. PQE60/PRE1 and pProEX-1/Gal80, the expression vectors forHis₆PRE and His₆Gal80, were provided by Prof. Stephen Johnston (UTSouthwestern, Dallas). pQE6O places the His₆ tag at the carboxy terminusof PRE1 while the His₆ tag for pProEX-1/Gal80 is at the N terminus ofGal80. The GST fusion of PRE1 was constructed by inserting a PCRamplicon containing the PRE1 gene into Nco1/BamHI-cut pGEXCSTEV plasmid(provided by Prof. Johnston). The plasmid expressing GST fused humanMdm2, pGEX Mdm2, was provided by Prof. David Lane, University of Dundee,Dundee. Construction of pMa1-c2X-Ndm2 was achieved by restriction digestof pGEX-Mdm2 with BamHI/EcoRI and ligation of the resulting fragmentinto the pMA1-c2X plasmid purchased from NEB. The plasmid encodingfull-length mouse Creb binding protein (CBP), was pRC/RSV-mCBP-HA-RK. Afragment of CBP including the KIX domain, amino acids 378-817, wasamplified by PCR and inserted into BamHI/HindIII digested pRSET-A vector(Invitrogen) to produce the resultant plasmid pRSET-CBP(378-8 17), whichexpresses the His₆ tag at the N-terminus of the protein. The plasmidexpressing the GST fusion of CBP(378-817), pGEX-2T-CBP(378-817), wasconstructed by insertion of the same CBP amplicon into BamHI/HindIIIdigested pGEX-2T vector purchased from Pharmacia Biotech that wasengineered with a HindIII site. A His₆ tag was added to the pGEX 2T-CBP(378-817) construct at the carboxy terminus of CBP by amplifying the CBPfragment with the primers 5′-CC GCG GGA TCC GCC TGT TCT CTC CCA CAC TGTCG-3′ (SEQ ID NO:1) and 5′-GAA TTC AAG CCT TTA GTG GTG ATG GTG GTG ATGGGC TGC TGG TTG CCC CAT GCC CAC AC-3′ (SEQ ID NO:2) and inserting backinto the BamHI/HindIII digested pGEX 2T vector.

Proteins and Antibodies. Glutathione-S-transferase (GST) fusion proteinswere expressed in E. coli cells gown in LB media+ampicillin to an OD₆₀₀of 0.6-0.8 and induced with 1 mM IPTG for 2-3 hours at 37° C. The cellswere lysed using sonication and centrifuged at 83,000 g to remove celldebris. Glutathione sepharose beads (Pharmacia Biotech) equilibratedwith PBS buffer were added to the lysate and incubated for 1 hour at 4°C. with agitation. The beads were poured into a column, washed with 20column volumes of PBS+0.5% triton X-100, followed by 10 column volumesof PBS buffer. The GST protein was eluted from the beads with 10 mMreduced glutathione in 50 mM tris, pH 8.0 and dialyzed into PBSbuffer+10% glycerol.

His₆ tagged fusions were expressed using the same protocol as describedabove for the GST fusions. After centrifugation, triton X-100 was addedto 1% Ni-NTA beads (Qiagen) that had been equilibrated with 50 mM sodiumphosphate buffer (PB)+300 mM NaCl were added to the lysate and incubatedfor one hour at 4° C. The beads were poured into a column, washed with20 column volumes of PB+500 mM NaCl+0.1% triton X-100+40 mM imidazolefollowed by 10 column volumes of 50 mM sodium phosphate buffer (PB)+300mM NaCl. The His₆ tagged protein was eluted from the beads with 400 mMimidazole and dialyzed into PBS buffer+10% glycerol.

MBP-Mdm2 was expressed in E. coli cells grown in rich broth (10 gtryptone, 5 g yeast extract, 5 g NaCl per liter)+glucose (2g/liter)+ampicillin to an OD₆₀₀ of 0.5 and induced with 0.3 mM IPTG for2 hours at 37° C. The cells were lysed using sonication and centrifugedat 87,000 g to remove cell debris. Amylose resin (NEB) equilibrated withcolumn buffer (20 mM tris+200 mM NaCl+1 mM EDTA) was added to the lysateand incubated for 1 hour at 4° C. The beads were poured into a columnand washed with 20 column volumes of column buffer. The MBP-Mdm2 waseluted from the beads with column buffer+10 mM maltose and dialyzed intoPBS buffer+10% glycerol.

All other proteins were obtained from commercial sources including VEGF(US Biological), MBP (NEB) and Glutathione-S-transferase (Sigma).Antibodies for western blotting were provided by Prof. Stephen Johnstonwith the exception of mouse anti-GST (sc-138) and goat anti-VEGF (sc-152G) which were purchased from Santa Cruz and mouse anti-Penta Histidinepurchased from Qiagen.

Synthesis of Peptides. Peptides were synthesized using a Symphonypeptide synthesizer (Protein Technology Incorporated) via Fmoc chemistryon Fmoc(aminoethyl)-Photolinker NovaSyn TentaGel resin (Nova Biochem).The linker is resistant to cleavage with TFA, therefore, the peptideside-chains can be deprotected with TFA without releasing the peptidefrom the bead. The bead-bound peptides were sequenced by automated EdmanDegradation on an Applied Biosystems 476A Protein Sequencer.

Fluorescently labeled peptides were synthesized using Fmoc chemistry onRink Amide MBHA resin (Nova Biochem). The peptides were modified at theN-terminus with 5(6)-carboxyfluorescein (Fluka) activated with HBTU(Advanced Chemtech). Peptides were cleaved from the resin with TFA andpurification was performed on a Biocad Sprint HPLC. The masses of eachpeptide were analyzed by MALDI-TOF mass spectrometry (Voyager DEPro—Applied Biosystems) and were within 0.1% of the predicted mass.

Phage Display. A 20 amino acid peptide library expressed at the Nterminal OH of M13 phage (named ON.543) was provided by Prof. StephenJohnston (originally obtained from Affymax (Needels et al., 1993) andcontained approximately 10⁸ different peptides. Pre1 binding peptide(PREbp) was isolated after six rounds of panning as follows. Round (RD)1: His₆PRE1(40 μg) was absorbed onto an ELISA plate, incubated with 10⁸phage in PBS (20 mM sodium phosphate, pH=7.5, and 150 mM NaCl) bufferfor 2 hours at room temperature, washed 8 times with 1×PBS+0.1% tween20, and eluted with 50 mM glycine HCl pH=2.0. Round (RD) 2: GST Pre1 (80μg) was bound to glutathione beads, incubated with 10⁸ phage from RD 1in PBS+0.5% triton X-100 for 2 hours at 4° C., washed 4 times PBS+1%triton X-100 then twice with PBS, and eluted by cleavage with TEVprotease. RD 3 GST Pre1 (40 μg) was bound to glutathione beads,incubated with 10⁹ phage from RD2 in PBS+1% triton X-100 for 2 hours at4° C., washed 4 times with PBS+1% triton X-100+350 mM NaCl, then twicewith PBS, and eluted by cleavage with TEV protease. RD 4 same as RD 3.RD 5 His₆PRE1(12 μg) was bound to Ni-NTA beads, incubated with 10¹⁰phage from RD4 in PBS+1% triton X-100+10 mM imidazole for 2 hours at 4°C., washed 4 times PBS+1% triton X+350 mM NaCl+20 mM imidazole, thentwice with PBS, and eluted with 200 mM imidazole. RD 6 same as RD 5.After round 6, phage DNA was isolated and sequenced. Since the freesynthetic peptide was insoluble, an additional six polar amino acidswere added, three on each end, to enable the peptide to be displayed onbeads.

CBP(378-817) binding peptide (KIXbp1) was found after four rounds ofpanning as follows. Round (RD) 1: GSTCBP(378-817) was absorbed onto anELISA plate, incubated with 10⁸ phage in PBS (20 mM sodium phosphate,pH=7.5, and 150 mM NaCl) buffer for 2 hours at room temperature, washed8 times with 1×PBS+0.1% tween 20, and eluted with 50 mM glycine HClpH=2.0. RD 2: GSTCBP(378-817) was bound to glutathione beads, incubatedwith 10⁹ phage from RD1 in PBS+1% triton X-100 for 2 hours at 4° C.,washed 4 times with PBS+1% triton X-100 then twice with PBS, and elutedwith 20 mM glutathione in 50 mM tris-HCl, pH=8.0. RD 3 same as RD2. RD 4GSTCBP(378-817) was bound to glutathione beads, incubated with 10⁹ phagefrom RD3 in PBS+1% triton X-100 for 2 hours at 4° C., washed 3 timeswith PBS+1% triton X-100+400 mM NaCl then twice with PBS, and elutedwith 20 mM glutathione in 50 mM tris-HCl, pH=8.0. After round 4, phageDNA was isolated and sequenced.

Determination of solution binding constants. Titration experiments weremonitored by fluorescence polarization spectroscopy using a PanVeraBeacon 2000 instrument capable of measuring anisotropy offluorescein-labeled molecules. Approximately 5 nM of thefluorescein-labeled peptide was added to 200 μL of PBS buffer (with theexception of VEGF; to conserve protein only 100 μL volume was used),which also contained 0.2 mg/ml bovine serum albumin and variousconcentrations of the target protein. The samples were incubated for 20minutes at room temperature to allow equilibrium to be reached. Thesample was then placed into the fluorescence spectrometer and thepolarization of the emitted light was measured. A plot of the change inanisotropy verses the protein concentration was used to determine thedissociation constant (K_(D)).

To determine the rate of dissociation of the peptide/protein complex insolution, the fluorescein-labeled peptide and the partner protein wereincubated at a protein concentration 10-fold above K_(D). Unlabeledpeptide was then added at a concentration 10- to 100-fold above thelabeled protein concentration. Fluorescence anisotropy measurements weretaken every 30 seconds. The anisotropy verses time was plotted and fitto a first order decay equation. In all cases, the dissociation reactionwas >90% complete in the time required to mix the unlabeled peptide withthe complex and return the cuvette to the spectrometer (approximately 10seconds).

Comparing Monomeric verses Dimeric Proteins. Approximately 0.02 g of p53peptide/tentagel was blocked with 10% milk overnight at 4° C. The beadswere split into 2 tubes and 6 μM of GSTMDM2 or MBPMDM2 was added alongwith 1% milk. The beads were incubated 2 hours at 4° C. and then washedfour times at room temperature for 5 minutes with 10 mM Tris(hydroxymethyl) aminomethane, pH=8.0, 150 mM NaCl, and 0.1% tween 20(TBST buffer) and then two times with 20 mM sodium phosphate, pH=7.5,and 150 mM NaCl (PBS buffer). After washing, each sample was aliquotedinto three tubes with excess buffer (15 mL PBS) added to tubes 2 and 3and exchanged every 15 minutes. The dissociation of the peptide/proteincomplexes was monitored at one hour and two hours. Gel loading-dye (50mM tris base, pH=6.8, 2% sodium dodecyl sulfate, 10% glycerol, 5%β-mercaptoethanol, 0.1% bromophenol blue, and 0.1% xylene cyanol) wasadded directly to the beads, which were then boiled. After cooling, thesupernatant was loaded onto a SDS (sodium dodecyl sulfate)polyacrylamide gel and analyzed by western blotting.

The same procedure above was carried out for GST-KIX and His₆KIX at aprotein concentration of 300 nM with the exception that BSA (3% forblocking and 0.3% for binding) was used instead of milk.

Dissociation of protein from peptide on Tentagel beads. Approximately0.02 g of peptide/Tentagel beads were blocked with 10% milk (exceptPRE1bp was blocked with 3% BSA) overnight at 4° C. One tenth of thebeads were aliquoted and set aside to be used as a control (tube #10).To the remaining beads, target protein (500 μL of a 300 nM solution) wasadded along with 1% milk (or 3% BSA+1% triton X-100 for PRE1) andallowed to bind for 2 hours at 4° C. The beads were then washed fourtimes at room temperature for 5 minutes with 10 mM Tris (hydroxymethyl)aminomethane, pH=8.0, 150 mM NaCl, and 0.1% tween 20 (TBST buffer) andthen two times with 20 mM phosphate, pH=7.5, and 150 mM NaCl (PBSbuffer). PRE1bp was washed four times at room temperature for 5 minuteswith 20 mM phosphate, pH=7.5, 350 mM NaCl, and 1% triton X-100 and thentwo times with PBS buffer. The beads were then equally divided into 9tubes and 10 or 15 mL of excess buffer was added to tubes 2-9. Tocontrol for possible rebinding of dissociated protein, to tube #10, theprotein was added to the peptide on beads after the addition of excessbuffer. The dissociation of the complex was monitored over a two-hourperiod with exchange of buffer every 15 minutes. Gel loading dye wasadded directly to the beads and after boiling, the supernatant wasloaded onto a SDS polyacrylamide gel and analyzed by western blotting.

Capturing Dilute Proteins. Approximately 0.014 g of Tentagel beadsdisplaying Gal80 binding peptide was blocked with 10% milk overnight at4° C. The beads were divided equally into 7 tubes and incubated with 500μL of buffer containing various amounts of His₆Gal80 protein (5 to 400ng) for 2 hours at 4° C. After washing four times with TBST buffer andtwice with PBS, gel loading dye was added directly to the beads and theamount of bound His₆Gal80 was determined as described above. A secondgel containing known amounts of pure His₆Gal80 was analyzedsimultaneously and was used as a standard to quantitate the amount ofHis₆Gal80 retained on the beads.

Lysate Experiment. E. coli lysate was prepared by growing BL21 cells toan OD₆₀₀ of 0.8. Cells were lysed by sonication in PBS buffer. Aftersonication, triton X-100 was added to 1%. His₆Gal80 was doped into theE. coli lysate (67 μg/mL) such that it constituted 5% of the totalprotein. Approximately 0.02 g of Tentagel beads on which theGal80-binding peptide had been synthesized and deprotected were added to300 μL of the E. coli lysate containing His₆Gal80 and incubated for 2hours at 4° C. The beads were washed four times for ten minutes eachtime at 4 with PBS buffer+1% triton X-100 at 150 mM NaCl at 4° C. andthen twice with PBS buffer. GSTGal4 AD (activation domain) waspre-incubated with glutathione sepharose beads and then used as apositive control to pull-down His₆Gal80 from the lysate under the sameconditions. Gel loading dye was added directly to the beads, loaded ontoa SDS polyacrylamide gel and analyzed by silver stain.

Example 2 Mixed Element Capture Agent Studies Results

Complex half life of immobilized peptides and dimeric proteins. Peptidelibraries are rich sources of protein-binding molecules and there existmany straightforward methods to screen such libraries. As mentioned inthe introduction, most such screens result in the isolation of ligandsthat form protein complexes with K_(D)s in the μM range. To test thesurface-mediated avidity concept, a collection of peptides known to binddifferent proteins were assembled, some homodimers and some monomers.Table 1 presents a list of equilibrium dissociation constants for thecomplexes used in this study. These values were determined by titratinga low level of fluorescein-labeled peptide with increasing amounts ofits protein partner and monitoring the level of binding by fluorescencepolarization spectroscopy (Heyduk et al., 1996). The values range from0.3 μM for the G80BPA/Gal80 protein complex (Han and Kodadek, 2000) to 8μM for the KIXBP1/KIX protein complex. To determine the rate ofdissociation of the peptide/protein complexes in solution, a largeexcess of unlabeled peptide was added to the protein/fluorescent peptidecomplex and the time-dependent decrease in polarization of thefluorescence was monitored. As expected from the modest equilibriumdissociation constants, the half-lives of all of these complexes wereall shorter than the time required to mix the solutions, which was about10 seconds (data not shown).

With these solution values in hand, the binding properties of the samepeptides immobilized on Tentagel beads were evaluated. Each peptide wassynthesized on Tentagel resin modified with an acid-stable linker,allowing deprotection of the peptide side chains without severing thelink to the bead. FIG. 6A shows the protocol that was employed toevaluate the kinetic stability of the immobilized peptide/proteincomplexes. The complex was formed by addition of excess protein to 18 mgof peptide-coated beads. After washing, the beads were divided equallyinto 9 tubes and then added to a large volume of buffer (10 or 15 ml),such that if the bound protein dissociated from the bead, it wouldpresumably be unable to re-associate due to its high dilution. Tofurther inhibit reassociation, the buffer was also changed every 15minutes to remove any free protein. The amount of protein remaining onthe beads was monitored at fifteen-minute intervals by denaturing gelelectrophoresis and Western blotting.

TABLE 1 Peptides employed in this study and the equilibrium dissociationconstants (K_(D)) of the peptide/protein complexes. Name ReferenceSequence K_(D)s (μM) Ga180bp Han and YDQDMQNNTFDDLFWKEGHR   0.3 Kodadek,2000 (SEQ ID NO: ID NO: 3) Ga180bpscram DLQRDTNKGFHEMFDWDYQN Nd (SEQ IDNO: ID NO: 4) Pre1bp SHSTARGEQERAAVYLWFTYDHRSER A (SEQ ID NO: ID NO: 5)Pre1bpscram SEFARDLAYGEYSQHVRWTHERATSR nd (SEQ ID NO: ID NO: 6) VEGFbpFairbrother et al., RGWVEICAADDYGRCLTEAQ >1^(b ) 1998 (SEQ ID NO: ID NO:7) VEGFbpscram CQECDYWREVRGADALITGA Nd (SEQ ID NO: ID NO: 8) Mdm2bpKussie et al., PLSQETFSDLWKLLPENNV 2 1996 (SEQ ID NO: ID NO: 9)Mdm2bpscram NVKWLDPNQELPSFLTSLE Nd (SEQ ID NO: ID NO: 10) KIXbp1SVPGSVSWFEFWSAVDAVET 8 (SEQ ID NO: ID NO: 11) KIXbp1scramFSASFTEVVDAGWVSPWSVE Nd (SEQ ID NO: ID NO: 12) Abbreviations: bp =binding peptide. Nd = not determined. scram = scrambled. The residuesshown in bold in the Prel by were added to enhance solubility. Allpeptide sequences are written with the N-terminus at the left end.^(a)Could not be determined due to limited solubility of the peptide.^(b)Lower limit. The true value could not be measured due to a limitedamount of protein. The peptide names indicate the protein theyrecognize.

FIG. 6B shows the results of experiments that employed the Gal80,GST-Pre1 and VEGF proteins, all native dimers, as well as dimericfusions of Mdm2 (GST-Mdm2) and KIX domain of Creb Binding Protein(GST-KIX). In the case of Gal80 protein the level of bound proteindecreased to approximately 50% of the original level within 30 minutesafter which the level of retained protein remained constant for at leastau additional one and a half hours. This biphasic behavior was expectedbased on the fact that the stable Gal80 dimers associate to formtetramers with a K_(D) in the mid to high nM range (Fancy and Kodadek1999; Melcher and Xu, 2001). Since the beads were originally exposed toexcess Gal80 at a protein concentration near or above the K_(D) of thetetramer, it seems likely that this form of the protein was the dominantspecies bound, but that only one of the component dimers was in contactwith the immobilized peptides. Thus, the inventors interpret theseresults as an initial dissociation of the Gal80 tetramer, leaving behinda tightly bound Gal80 dimer that does not dissociate from the beadsduring the course of the experiment. GST-Pre1, VEGF, GST-KIX andGST-Mdm2 were also bound very stably to the beads. Little, if anydissociation of these dimeric proteins was observed over the course oftwo hours. To ensure that these results truly represented stable,specific peptide/protein complexes, several controls were performed.Lane 10 in FIG. 6B shows the result of adding an amount of protein equalto the “t=0” level to the highly dilute bead+buffer mixture andincubating for two hours. In accordance to the procedure mentionedabove, the buffer was exchange every fifteen minutes to discourageassociation of the free protein to the peptide immobilized on Tentagel.This control was done to assess whether the protein could re-associatewith the beads in the diluted sample if it dissociated. No associationof the VEGF, GST-Pre1, GST-KIX or GST-Mdm2 was observed under theseconditions and only a trace of Gal80 was present. The experiment wasalso repeated with beads displaying a scrambled version of the bindingpeptide (see Table 1) and beads lacking any peptide. In each case, nobound protein was observed with the exception of a small amount ofbinding of the Pre1 protein to the scramble peptide that presumablyrepresents a minor non-specific interaction. The half-lives of theseimmobilized peptide/protein complexes are several hours or more, anincrease of at least three orders of magnitude compared to that measuredin solution.

Kinetic stability of immobilized peptides with analogous monomeric anddimeric proteins. It seems likely that the huge difference between thekinetic stabilities of these peptide/protein complexes in solution andon beads is due to bidentate binding of the immobilized peptides to thedimeric proteins. However, other possibilities cannot be ruled out basedon these data alone. To probe this issue further, similar experimentswere carried out using dimeric and monomeric versions of the sameprotein.

The domain of the human Mdm2 protein represented by residues 1-188 is astructurally characterized monomer. This Mdm2 fragment is known to binda peptide derived from the p53 activation domain (Kussie et al., 1996.).Two different Mdm2-containing fusion proteins were expressed andpurified. In one, the Mdm2 domain was fused to maltose-binding protein(MBP), a monomer. In the other it was fused to GST, a native dimer.Titration experiments showed that, in solution, the p53 derived peptidebound each form of the protein with similar affinity and kinetics (K_(D)approx. 2 μM (Table 1) with a half life of less than 10 seconds). Thep53-derived peptide had no detectable affinity for GST or MBP alone(data not shown).

The kinetic stabilities of each of these complexes were then probed withthe peptide immobilized on Tentagel beads, using a dilution protocolsimilar to that shown in FIG. 6A, except that fewer time points weretaken. As shown in FIG. 8A (lanes 6-9), monomeric MBP-Mdm2 dissociatedfrom the beads rapidly. Only a small fraction of the input protein wasdetectable immediately after completion of the washing steps (lane 7)and no trace of protein was detectable on the beads after a one hourincubation (lane 8). In stark contrast, little or no dissociation of thedimeric GST-Mdm2 fusion protein was observed even after two hours.Again, a dilution control demonstrated that if the protein haddissociated from the beads under these conditions, reassociation wouldnot have occurred.

Similar experiments were also carried out with monomeric and dimericforms of the KIX domain (Radhakrishnan et al., 1997) of the CRES-bindingprotein (CBP), a transcriptional coactivator. In this case, the peptideemployed was isolated by phage display from a library of 20mers (seeMaterials and Methods). The peptide/protein complexes (including eithermonomeric His₆-KIX or dimeric GST-KIX) exhibited K_(D)s of approximately8 μM in solution (Table 1). However, the half-lives of the twopeptide-protein complexes differed greatly when the peptide wasimmobilized on Tentagel. FIG. 8B (lanes 2 and 4) displays the amount ofprotein remaining on the beads immediately after the washing steps.Monomeric His₆-KIX was undetectable whereas dimeric GST-KIX was present.As was shown in FIG. 6, GST-KIX and immobilized KIXbp1 possessed ahalf-life in excess of two hours.

The striking differences between the half-lives of complexes containingmonomeric and dimeric forms of the same proteins on the peptide-coatedTentagel beads strongly supports the idea that stable binding of nativedimers is due to bidentate binding.

Efficient capture of dilute proteins. In all of the above examples, theprotein was loaded onto the peptide-coated bead at a relatively highconcentration (300 nM) prior to dilution. Since the concentration ofmost proteins of interest in a biological sample will be lower, it wasof interest to evaluate the ability of a Tentagel-bound peptide tocapture a dimeric protein from more dilute solutions. FIG. 7 shows theresults of a study in which 2 mg of Gal80 bp-coated Tentagel beads(approximately 0.4 μmoles of peptide) was incubated with the indicatedamounts Gal80 protein in a volume of 500 μl. After washing, the boundprotein was detected by boiling the beads in denaturing loading bufferfollowed by SDS-PAGE/western blot analysis of the resulting supernatant.The bottom panel in FIG. 7 shows a western blot in which known amountsof purified Gal80 were applied to the gel, allowing quantitation of theamount of protein retained by the bead-bound peptide in the experiment.The results show that a constant amount of Gal80 protein was captured bythe beads at protein concentrations ranging from 40 nM to 0.4 nM. Evenat the lowest protein concentration detectable, essentially 100% of theprotein was bound to the beads, as can be seen by comparing the bandintensities in the experimental and calibration blots. Further dilutionof the protein in this assay format exceeded the sensitivity of thewestern blot. This suggests that the K_(D) of the immobilizedpeptide/Gal80 complex must be at least an order of magnitude lower than0.4 nM. At all of the higher protein concentrations, a constant amountof Gal80 protein (approximately 50 ng) was retained on the beads. Thismust reflect the binding capacity of the beads.

Specificity of the peptide-protein interaction. In a protein-detectingarray, a complex solution such as a blood sample or cell extractcontaining thousands of proteins would be applied to the array. Forproper interpretation of the results, it is critical that thespecificity of binding of the target protein to its cognate captureagent is high.

To address this point, the Gal80 bp/Gal80 pair was again employed.His₆Gal80 was doped into an E. coli lysate such that Gal80 represented5% of the total protein concentration. This solution was then incubatedwith the Tentagel-bound Gal80 bp followed by thorough washing to removeany unbound material. The composition of the proteins captured by thepeptide was then addressed by SDS-PAGE followed by silver staining. Asshown in FIG. 5, Gal80 was the only detectable protein retained from theextract, at least at the level detectable by silver staining. Nodetectable proteins were retained in a control experiment using ascrambled version of Gal80 bp. To provide context for this result, thesame Gal80-doped extract was applied to glutathione-sepharose-boundGST-Gal4 AD. This 34 residue fragment of Gal4 is the native ligand forGal80 and is known to form a high affinity and specificity complex withthe repressor (Johnston et al., 1987). As can be seen by comparing lanesin FIG. 5, the results obtained using the peptide and the native Ga14 ADwere quite similar (the large doublet of bands near the bottom of thegel is due to the GST-Ga14 AD fusion protein that was eluted from thebeads).

Efficient capture of a monomeric, two domain model protein. FIGS. 9A-9Bshow that the MECA concept can be applied to a monomeric protein (asdemonstrated by gel filtration; data not shown). Peptides that bind MBPand human mdm2 protein were identified. Each peptide/protein complex wasfound to have a K_(D) in the μM range and both peptides bound a MBP-Mdm2fusion protein with the same modest affinities (FIG. 9B). Bothindividual peptides bind the Texas Red-labeled fusion protein poorlyunder demanding conditions. However, a MECA comprised of the peptidesfused together via a single intervening serine residue (a chimericbinding element) bound the protein efficiently (see FIG. 9A for afluorescent micrograph of the two bead mixtures, showing the level ofcontrast). However, the MECA did not bind the fusion protein tightly insolution (FIG. 9B) exhibiting less than two-fold improvement over theindividual peptides. This proves that the high affinity binding on thesurface must have involved more than one immobilized molecule and thefusion protein (FIG. 10). In further support of this notion, the numberof serines placed between the binding elements has no effect on theaffinity of the MECA on the surface (data not shown.)

Example 3 Peptoid Library Studies Materials and Methods

Reagents and Instrumentation. All reagents and solvents were purchasedfrom commercial suppliers and used without further purification.TentaGel macrobeads (140-170 micron diameter; substitution: 0.51 mmol/g)were obtained from Rapp Polymere (Tübingen, Germany). Analytical HPLCwas performed on a Biocad Sprint system with a C18 reverse-phase HPLCcolumn (Vydac, Columbia, Md., 5 μM, 4.6 mm i.d.×250 mm). A gradientelution of 10-50% B in 20 minutes followed by 50-80% B in 5 minutes wasused at a flow rate of 1 mL/min. (solvent A: H₂O/0.1% TFA; solvent B:CH₃CN/0.1% TFA). MALDI-TOF MS was performed on a Voyager-DE PRObiospectrometry workstation (Applied Biosystems, Foster City, Calif.)using α-hydroxy cinnamic acid as the matrix. A New Brunswick Scientific(Edison, N.J.) Innova 4400 incubator shaker was used to perform thepeptoid syntheses at 37° C. Microwave-assisted peptoid syntheses wereperformed on a 1000 W Whirlpool (Benton Harbor, Mich.) microwave oven(model MT1130SG) set to deliver 10% power. Edman sequencing of peptoidswas performed on an ABI 476A Protein Sequencer (Applied Biosystems,Foster City, Calif.). The fluorescence spectra of the beads wererecorded with a hyperspectral microscope constructed in the laboratoryof Professor Harold Garner (UT-Southwestern) (Schultz et al., 2001). Theon-bead fluorescence assays were visualized with a Nikon Eclipse TE300fluorescence microscope equipped with a Chroma 61002 triple band filterset and a CCD camera (Preston, United Kingdom). MetaMorph software(Universal Imaging Corp., Downingtown, Pa.) was used to acquire andprocess the photomicrographs. Isothermal titration calorimetry (ITC)experiments were performed on a MicroCal VP-ITC instrument (Northampton,Mass.).

Syntheses of peptoid libraries at 37° C. The syntheses of 8-merlibraries were performed in standard 25 ml glass peptide synthesisreaction vessels (Chemglass, Vineland, N.J.) in an incubator shaker at37° C. One and a half grams of TentaGel macrobeads (140-170 μm;substitution: 0.51 mmol/g) were distributed equally into 5 peptidesynthesis reaction vessels, 5 ml of DMF was added and the beads wereallowed to swell at room temperature for 60 minutes. The DMF was drainedand 1.5 ml of 2M bromoacetic acid and 1.5 ml of 3.2Mdiisopropylcarbodiimide (DIC) was added to each vessel. The reactionvessels were placed on an incubator shaker set at 37° C. and 225 rpm for40 minutes. The vessels were drained and the beads were thoroughlywashed with DMF (8×3 ml). The beads in each of the vessels were treatedwith one of five amines (see FIG. 13) at 2M concentration and allowed toreact in the shaker at 37° C. for 60 minutes. All the amines weredissolved in DMF, except 4-(2-aminoethyl)benzene sulfonamide which wasdissolved in DMSO. The vessels were drained and washed thoroughly withDMF (8×3 ml). The beads in each of the reaction vessels were pooled intoa large 250 ml peptide synthesis vessel, drained, suspended in 50 ml ofdichloromethane/DMF (2:1) and randomized by bubbling argon for 15minutes. The beads were distributed equally into five 25 ml peptidesynthesis vessels and the procedure was repeated. The protocol wasslightly modified for the final 4 residues of the library, where thedisplacement of the bromide by the primary amine was carried out for 90minutes, instead of 60 minutes. In the case of the 78,125 compoundlibrary, the fourth residue from the amino terminus was fixed and thus,all the reaction vessels were treated with ethanolamine for the bromidedisplacement step.

Microwave-assisted peptoid library syntheses. The syntheses of 5-mer and6-mer libraries were performed employing a microwave-assisted protocol(Olivos et al., 2002) on 1 g and 2 g of beads, respectively. In thisprotocol, both the acylation and bromide displacement by the primaryamine were performed twice for 15 seconds in a 1000 W microwave oven setto deliver 10% power. The beads were shaken manually for 30 secondsbetween microwave pulses to ensure proper mixing. All other steps wereidentical to the 37° C. procedure.

Protection and deprotection in primary amines. The functional groups inamine 1 (hydroxy), amine 3 (primary amino) and amine 7 (secondary amino)were protected following previously reported literature procedures (Unoet al., 1999; Pons et al., 1998). The following procedure was used tocleave the protecting groups at the end of the library synthesis. Thebeads were washed thoroughly with DMF (8×3 ml) then dichloromethane (3×3ml), drained and treated with 6 mL of 95% TFA, 2.5% water and 2.5%anisole for 2 hours. The cleavage cocktail was drained and the beadswere washed thoroughly with dichloromethane (8×3 ml). The beads wereneutralized by treating with 10% diisopropylethyl amine in DMF for 5minutes, washed with dichloromethane (5×3 ml), and dried until furtheruse.

For re-synthesis and characterization of peptoids by HPLC and MALDI-TOF,syntheses were performed on 50 mg of Fmoc-Rink amide MHBA resin(substitution: 0.73 mmol/g; Nova Biochem, Laeufelfingen Switzerland).The beads were swollen in DMF for 30 minutes, drained, treated twicewith 20% piperidine in DMF for 10 minutes (2×2 ml) and washed with DMF(8×3 mL). The peptoid sequence was constructed by the microwave-assistedprotocol (Chene et al., 2000) and washed thoroughly with DMF (8×3 mL)and dichloromethane (3×3 mL). The peptoid was released from the resinwith concomitant removal of protecting groups by treating the beads with6 mL of 95% TFA, 2.5% water and 2.5% anisole for 2 hours. The suspensionwas filtered and the filtrate concentrated by blowing nitrogen over thesolution. The concentrated filtrate was dissolved in 2 mL of 1:1acetonitrile/water and lyophilized. The resultant solid was subjected toHPLC and MALDI-TOF analysis.

Sequencing peptoids by Edman degradation. The sequencing of peptoids wasperformed on an ABI 476A protein sequencer, using the FSTNML program anda standard gradient (Gradient 1). The FSTNML program was slightlymodified by adding a 60 second “wait” step at the end of the cycle toenable the gradient to run slightly longer than normal.

Protein purification. Glutathione-S-Transferase (GST) was expressed inE. coli BL21-RIL from the commercially available plasmid pGEX-2T(Amersham Biosciences, Piscataway, N.J.). The cells were grown until anOD₆₀₀ of 0.8 was reached, at which time 1 mM IPTG was added to themedium to induce protein expression. After further growth at 37° C. for3 hrs, the cells were harvested, sonicated and centrifuged at 22,000rpm. The cleared lysate was then incubated with glutathione-agarosebeads equilibrated with PBS at 4° C. for 1 hr. The beads were washedwith 10-12 volumes of PBS, packed into a column and further rinsed withPBS. GST bound to the beads was eluted with 10 mM reducedglutathione/PBS and fractions were collected and analyzed on a 12%denaturing polyacrylamide gel. The fractions containing highly purifiedGST were pooled and dialyzed against PBS+10% glycerol. The proteinconcentration was estimated using Coomassie Plus Protein Assay ReagentKit using BSA as a standard. MBP-mdm2 was overexpressed from pMAL-mdm2in BL21-RIL cells (Bachawat-Sikder and Kadadek; 2003). Herein, theconditions were slightly modified; cells were grown in the presence of0.2% glucose and induced at OD_(600nm)=0.5 with 0.3 mM IPTG and grownfor an additional 3 hrs. A buffer consisting of 20 mM Tris-HC1+200 mMNaCl+1 mM EDTA, pH 7.4 was used. The protein was bound to amylose resinand after thorough washing, was eluted with 10 mM maltose.

Protein labeling with Texas Red. The protein solution (preferably 2mg/ml) was adjusted to pH 8.3 with 0.2 M NaHCO₃ buffer. To this 5 μl of50 mg/ml Texas Red solution in DMF was added with mild vortexing to mixthe sample. This solution was incubated with tumbling at roomtemperature for one hour, after which the reaction was quenched with 1.5M hydroxylamine. Dye-conjugated protein was separated from excess dyeusing a desalting column. Measurement of the absorbance of the sample at280 nm and 595 nm indicated that, on average, these conditions resultedin each protein molecule acquiring one molecule of Texas Red.

Preparation of E. Coli lysate for screening studies. The E. coli(BL21-RIL strain) cells were grown overnight at 37° C. in Luria broth.The cells were harvested by low speed centrifugation, washed andresuspended in sonication buffer (50 mM NaH₂PO₄ pH 8.0, 300 mM NaCl,0.1% Tween 20+protease inhibitor). The cells were then sonicated andcentrifuged at 22,000 rpm to remove cell debris and provide the clearedcell lysate. The concentration of the lysate was estimated using theBradford assay with BSA as a standard.

Library screening and identification of hits. TentaGel beads (150 mg;approx. 78,000 beads) harboring the combinatorial library X₃-Nser-X₄were swollen in TBST (50 mM Tris pH 7.4, 150 mM NaCl, 0.1% Tween 20) for1 hour, after which they were blocked with E. coli lysate at roomtemperature for one hour. The lysate was removed and the beads wereincubated with 50 nM Texas Red-conjugated MBP-Mdm2 in TBST containing 1M NaCl+1% Tween-20, in the presence of a 1000-fold excess of E. colilysate (assuming the average molecular mass of the proteins in thelysate to be the same as of the target protein), for one hour at roomtemperature. The beads were washed with TBST (6×1 mL) and visualizedunder a fluorescence microscope fitted with a Texas Red filter. Thebrightest beads were isolated manually with a pipette tip. In anotherexperiment, 100 mg of library X₅ was screened for GST binding peptoidligands. The beads were blocked with 5% milk/TBST and then incubatedwith 1 μM Texas Red-labeled GST in the presence of 1000-fold excess ofE. coli lysate.

After picking the putative “hits,” each bead was heated in a 1% SDSsolution for 20 minutes, followed by three washes with 1×PBS. They werethen sequenced by Edman degradation.

Isothermal titration calorimetry (ITC). ITC experiments were conductedon a MicroCal VP-ITC instrument. For the titration, 70 μM MBP-hmdm2 or30 μM GST in PBS+10% DMSO was taken in the sample cell. To this, 15 μlaliquots of the peptoid solution in the same buffer were added from acomputer-controlled 250 μl rotating syringe. The syringe was set at 400rpm with intervals of 3 minutes between injections to attain baselinestabilization. The heat absorbed or released accompanying the titrationwas recorded as differential power (DP) by the instrument software.Experiments were carried out with C values between 1 and 400. The totalheat recorded was then fitted via a non-linear least-squaresminimization method. Titration of the ligand solution with the bufferalone gave the heats of dilution. Titration with MBP alone was recordedunder identical conditions.

Protein capture assays using TentaGel-displayed peptoids. Fivemilligrams of TentaGel beads (displaying the respective hit sequences,Nlys-Nbsa-Nlys-Nser-Nbsa-Npip-Nbsa-Npip-CONH₂ andNbsa-Nlys-Nbsa-Npip-Nlys-CONH₂ or a random sequenceNpip-Nser-Nbsa-Nall-Nlys-Npip-CONH₂) were equilibrated inphosphate-buffered saline (PBS) for 60 minutes. The buffer was removedand the beads were blocked with 2% BSA for 60 minutes to saturate anynon-specific binding sites. The beads were then washed with PBS (3times), and incubated with 500 nM (unless indicated otherwise) of aTexas Red-labeled protein (MBP-MDM2 or GST) in 2% BSA (in 1×TBSTbuffer), in a 300 μL volume for 60 minutes. The beads were washed withTBST six times to remove any unbound protein and photographed under afluorescence microscope. Studies that employed native (unlabeled)proteins were performed as follows. Ten milligrams of beads displayingthe peptoid were exposed to 1 μM protein in the presence of 1000-foldexcess E. coli lysate, 0.2% Tween-20 and 0.2 M NaCl in a total volume of2 ml at RT for 2 hrs. The beads were washed three times with TBST (20 mMTris buffered saline+0.1% Tween-20). Ten microliters of 2×SDS-PAGEloading dye was then added directly to these beads and they were boiledfor 10 mins. The entire supernatant was loaded onto a 12% denaturingpolyacrylamide gel and analyzed by Western blot using anti-Mdm2antibodies for MBP-Mdm2 and anti-GST antibodies for GST.

Dilution experiment. Fifteen milligrams TentaGel beads displayingNbsa-Nlys-Nbsa-Npip-Nlys-CONH₂ were equilibrated in PBS for 60 min. Thebuffer was removed and the beads were incubated with E. coli lysate for60 minutes to block any non-specific binding sites. The beads werewashed with PBS three times and split into three Eppendorf tubes. Thebeads were incubated with 1 μM, 500 nM or 100 nM respectively, of TexasRed-labeled GST in the presence of a 100-fold excess of E. coli lysatein a 300 volume for 60 minutes. The beads were washed with TBST sixtimes to remove any unbound protein and visualized under a fluorescencemicroscope. The experiment was also done at 10 nM protein, but little orno fluorescence above background was observed (not shown).

Example 4 Peptoid Library Studies Results

Synthesis and characterization of a peptoid library. One goal was toconstruct a peptoid library of many thousands of compounds. As a firststep, some new amines were confirmed as good building blocks (forexample compounds 2, 5, 8 and 11 in FIG. 12). Various other amines maywork well in peptoid synthesis including, but not limited to the aminesillustrated in FIG. 12 (Kirshenbaum et al., 1998; Zuckerman et al.,1994; Figliozzi et al., 1996; Wender et al., 2000; Burkoth et al.,2002). The exemplary amines shown in FIG. 12 were found to provideexcellent yields in the sub-monomer protocol (FIG. 21). In each case,this was determined by the synthesis of a test pentameric peptoid inwhich the amine in question was used in steps two and four and residues1, 3 and 5 were derived from the well-characterized benzylamine(Figliozzi et al., 1996). In each case, the desired pentamer wasisolated in at least a 85% yield.

One aspect of the invention is the identification of compounds orbinding elements capable of capturing proteins, i.e., identifying acapture agent, when immobilized on arrays. In one embodiment, theinventors screen libraries on a resin, rather than physicallysegregating beads and releasing the compound into solution as isgenerally done for chemical genetics (Clemons et al., 2001; Blackwell etal., 2001). Thus, in certain embodiments, the inventors preferred aresin with: 1) good swelling properties in organic solvents and in waterto support both efficient synthesis and to provide ready access of thebound peptoids to proteins in aqueous buffer, 2) a sufficiently highloading capacity that the structure of “hits” from a screen can bedetermined unambiguously by direct Edman or mass spectrometry (MS)-basedsequencing, eliminating the need for encoding, 3) a low fluorescencebackground, since the screening experiments will most conveniently becarried out with fluorescently labeled protein. PEGA, a polyacrylamidebased resin, which is employed by several workers in the combinatorialchemistry field, generally satisfied these criteria. However, thesebeads are extremely fragile mechanically and this introduced somedifficulty in the screening studies due to the large number of brokenbeads that were present in any given library. Therefore, the inventorspreferred to employ a more stable polystyrene-based bead. Afterassessment of various options, TentaGel-Macrobeads (140-170 μm indiameter from Rapp Polymere) were selected as the preferred resin. Whileit has a hydrophobic core, the TentaGel resin is derivatized withpolyethylene glycol chains that not only greatly improve the swelling ofthe beads in aqueous solution, but also provide a “non-sticky” surfacethat is ideal for reducing non-specific protein binding duringscreening. TentaGel beads do demonstrate an intrinsic fluorescence,which complicates screening, but is tolerable.

The initial library employed amines 1-5 of FIG. 12 and had the generalformula X₃-Nser-X₄, where X represents any of the monomers derived fromamines 1-5 of FIG. 12. A standard split and pool synthesis scheme (Lamet al., 1991) using 1.5 g of beads was employed to create thecombinatorial library, which has a theoretical diversity of 78,125compounds. The protocol employed to create the first four residues was aslight modification of the published sub-monomer procedure (Figliozzi etal., 1996; Kirshenbaum et al., 1998) in which the acylation step wascarried out with 2 M bromoacetic acid and 3.2 M diisopropylcarbodiimide(DIC) in DMF for 40 minutes at 37° C. followed by displacement of thebromide with 2 M primary amine for one hour. For the subsequentresidues, the amine addition step was allowed to proceed for 90 minutesat 37° C. All primary amines were dissolved in DMF, except4-(2-aminoethyl)benzene sulfonamide, which was dissolved in DMSO. Theresin was pooled into a 250 ml glass peptide synthesis reaction vessel,mixed by bubbling argon through the suspension for 15 minutes and splitbefore each acylation step. At the end of the synthesis, the side chainprotecting groups, if present, were removed by treating with 95% TFA,2.5% water and 2.5% anisole for 2 hours. The resin was then neutralizedwith 10% DIEA in DMF, washed with DCM and dried until further use.

To determine the quality of the library, several tests were conducted.Unfortunately, the amount of compound present on a single bead is toosmall to allow direct characterization by HPLC or spectroscopic means,but to further address the likely purity of the library members, an8-mer peptoid was synthesized on Rink amide MHBA resin using amines 1-5of FIG. 12 (sequence: Nser-Nlys-Nall-Nlys-Nbsa-Npip-Nbsa-Npip-CONH₂).The final product was released from the beads using 95% TFA, 2.5% water,2.5% anisole and the material was characterized by HPLC and massspectrometry. Data indicated that the major peak in the HPLCcorresponded to the expected compound. While this experiment cannotaccount for potential context-dependent effects in the synthesis of acombinatorial library, it does demonstrate that all of the monomers workwell in the synthesis, consistent with the previous tests of eachmonomer using the benzyl amine assay described above.

To evaluate diversity, ten beads from the library were chosen at randomand the displayed peptoids were sequenced by automated Edmandegradation. Boeijen and Liskamp (1998) reported that peptoids can besequenced by Edman chemistry using several beads as the input. However,for library screening studies, the ability to sequence a single bead ispreferred. An automated approach would be even more advantageous, as itwould eliminate the practical difficulties involved in handlingindividual beads for long periods of time, over several cycles ofchemistry. A commercial peptide sequencer (ABI 476A) was adapted for thesequencing of peptoids. Although larger (400-500 μm) TentaGel macrobeadsare available commercially that allow spectroscopic analysis of thecompounds derived from a single bead, such beads introduce practicallimitations on the size of the libraries that can be constructed andhence, were not employed in this study. The typical HPLC protocol usedfor sequencing peptides was modified slightly to allow the gradient torun longer. As shown in FIG. 13, when ten beads were picked from thelibrary and subjected to Edman degradation, the derived sequence of eachpeptoid was different, as expected for a large, diverse library. Thechromatographic traces from these sequencing runs also showed that eachof the peptoids was full-length. At each step of the Edman process, onlyone major peak was generally observed, with the exception of a smallamount of signal resulting from the previous and subsequent monomers inthe peptoid, which is commonly observed in peptide sequencing using thischemistry.

Isolation of Mdm2-binding peptoids from the library. With a high qualitypeptoid library in hand, the inventors developed appropriate conditionsfor on-bead screening. To facilitate future efforts to automatescreening using a fluorescence-activated bead sorter fluorescentlylabeled proteins were to be employed in screening. However, TentaGelresin had an intrinsic fluorescence, particularly in the green region ofthe spectrum (FIG. 14). This “background fluorescence” rendered the useof many organic dyes, such as fluorescein, impractical for screeningwith TentaGel resin. However, the intensity of the bead fluorescencedropped significantly in the red region of the spectrum. Thus, TexasRed-labeled proteins were evaluated as potential targets in thescreening process.

The human Mdm2 protein is a negative regulator of p53 function and apotential anti-cancer drug target. A fragment of Mdm2 (residues 1-1 88)was fused to maltose-binding protein (MBP) and used as an initialtarget, since this fusion protein expressed at higher levels than theMdm2 fragment alone. This region of Mdm2 includes the region of thenative protein that binds the p53 activation domain (Kussie et al.,1996) and there have been several previous reports of isolation of apeptide or small molecule ligands for this region of Mdm2 (Stoll et al.,2001; Bottger et al., 1997; Chene et al., 2000). Thus, the inventorssuspected that this protein would represent a reasonable target for theinitial peptoid library screening studies.

Detailed screening conditions are describe above. In general, it wasfound to be optimal to employ challenging conditions in order toeliminate low affinity or low specificity hits. For example, the use ofa high salt- and detergent-containing buffers (1M NaCl and 1% Tween-20)are preferred. The concentration of the Texas Red-labeled protein wasonly 50 nM since studies conducted at higher protein concentrationsindicated that a larger fraction of the library registered as “hits,”presumably representing weaker ligands. A 1,000-fold excess (based onmass) of cleared E. coli lysate was used in order to demand highspecificity. Screening experiments that employed only a singlecompetitor protein such as bovine serum albumin (BSA) provided poorerresults.

After incubating the labeled maltose-binding protein MBP-Mdm2 fusionprotein with the bead library for one hour under these conditions andthen washing the beads six times with the same buffer, beads thatexhibited above background fluorescence were identified visually using afluorescence microscope. FIG. 15 shows a photomicrograph of a fieldcontaining a bead that was scored as a hit. This bead (marked with thearrow in the figure) is clearly brighter than the surrounding beads, butthat all of the others are far from dark. This is, in part, a reflectionof the intrinsic fluorescence of the beads (FIG. 14) as well as the lowlevel, non-specific binding of some of the labeled protein to many beadsin the library, despite the presence of high levels of competitor.Fortunately, while this background is annoying and reduces the speed atwhich libraries can be screened visually, it is tolerable. Eleven hits(approximately 0.014% of the input beads) with a fluorescence well abovebackground were identified.

To identify the sequence of each peptoid hit, bright beads were pickedmanually using a pipette. The individual beads were then heated to 95°C. in 1% SDS and placed in the chamber of an automated Edman sequencer.Some consensus was observed among the hits at positions 1, 2 and 8. TheEdman sequencing trace of the brightest bead among the eleven hits isshown in FIG. 15B, which clearly identified its sequence (FIG. 15C).

Validation of the putative Mdm2 ligands. An issue in any libraryscreening experiment is to validate the resynthesized ligands. Beadlibrary-derived ligands often fail in typical solution binding assaysfor many reasons. For example, avidity or context effects unique to thesolid surface on which the library was constructed could allow it towork well on resin but behave poorly in solution binding assays. Evenmore problematic is the possibility that the true ligand might have beena minor component on the bead due to some sort of side reaction duringthe synthesis and is not the compound expected from the sequencing data.

The putative hit (see FIG. 15C) was resynthesized on Rink resin, cleavedand purified to apparent homogeneity by HPLC. Binding of the syntheticpeptoid to MBP-Mdm2 was then analyzed by isothermal titrationcalorimetry (ITC) (Leavitt and Freire, 2001). The data (see FIG. 16A)indicated an equilibrium dissociation constant of 37 μM. When thetitration experiment was repeated with MBP, little or no binding wasobserved (FIG. 16B). This observation both suggests that the peptoidligand isolated is specific and that it recognizes the Mdm2-deriveddomain of the MBP-Mdm2 fusion protein against which it had beenselected.

Given the goal of constructing protein-detecting microarrays based onpeptoids or other synthetic compounds, it was of even greater interestto us to determine the binding properties of the resynthesized compoundwhen affixed to a solid surface. To this end the experiments shown inFIG. 17 were carried out. First, the hit was resynthesized on TentaGeland the protecting groups removed without removing the peptoid from thebead. It was then incubated with either Texas Red-labeled MBP-Mdm2 orTexas Red-labeled MBP at the protein concentration indicated in thefigure in the presence of 2% bovine serum albumin (BSA) as competitor.As shown in FIG. 17A, the beads captured the MBP-Mdm2 proteinefficiently, while little MBP binding was observed. This corroboratesthe ITC data. To eliminate the possibility that the Texas Red labelcontributes significantly to binding of the protein to the immobilizedpeptoid, a study was conducted using native MBP-Mdm2. Unlabeled proteinwas incubated with the peptoid hit on TentaGel beads in the presence ofa 1000-fold excess of E. coli proteins. The beads were then pelleted andwashed. As shown in FIG. 17B, lane 3, SDS-PAGE/Western blot analysisrevealed that the immobilized peptoid had retained about 10% of theMBP-Mdm2 protein present (note that the protein was present in molarexcess over the peptoid, so complete retention of the input was notpossible). No detectable MBP-Mdm2 protein was retained when this studywas repeated with a random peptoid, (lane 4) or TentaGel beads lacking adisplayed peptoid (lane 5). The peptoid selected in the library screenis an Mdm2-binding compound capable of capturing the protein fromcomplex mixtures such as model cell extracts.

Larger, chemically diverse peptoid libraries. The inventors haveconstructed larger and/or more chemically diverse libraries to supportfuture larger scale screening against various other protein targets. Asbefore, standard split/pool synthesis (Lam et al., 1991) on TentaGel wasemployed. The first library utilized amines 3-6 and 8-13 of FIG. 12 andconsisted of five residue peptoids, providing a theoretical diversity of100,000 compounds. While not much larger than the 78,125 member librarydiscussed above, this library is far more diverse chemically, since 10different amines were employed in its construction, with each positionrandomized. The second library employed only five monomers, but waslonger, consisting of randomized octamers, providing a theoreticaldiversity of 390,625 compounds. Finally, an extremely large library ofrandomized hexamers was made using nine different amines, providing atheoretical diversity of 531,441 compounds.

All of these libraries were characterized for quality in the same way asdescribed above for the 78,125 compound library. Some of the data forthe largest of the libraries are presented in FIG. 18A. FIG. 18A showsthe results of sequencing 10 beads chosen at random from the library. Asexpected, all were different. The Edman traces again suggested thatfull-length peptoids were obtained in each case (FIG. 18B). Two mixedsequence hexamers, Ntrp-Nmea-Npip-Nlys-Nffa-Nmba-CONH₂ andNbsa-Nleu-Napp-Nffa-Nmea-Npip-CONH₂, were synthesized and shown by HPLCto be >85% pure. Between them, these hexamers contain all of themonomers that were subsequently employed in the library construction.The results again suggest that in the absence of unexpected contexteffects, all of the coupling steps proceed in high yield.

To determine if these libraries would be facile sources of proteinligands or binding elements as well, part of the 100,000 compoundlibrary was screened against Texas Red-labeled GST using conditionssimilar to those described above except that lower salt and detergentconcentrations were employed. Of the approximately 50,000 beads used inthis screen, (0.5% of total population) displayed red fluorescence wellabove the background. One of the brightest beads was picked and thepeptoid sequence determined by Edman degradation to beNbsa-Nlys-Nbsa-Npip-Nlys-CONH₂ (FIG. 19). Since the primary interest isin evaluating the ligands or binding elements isolated from thesescreens for their ability to retain proteins from biological sampleswhen attached to a surface, a number of on bead assays were conductedwith Nbsa-Nlys-Nbsa-Npip-Nlys-CONH₂ (FIG. 20). As shown in FIG. 20A, theresynthesized compound retained Texas Red-labeled GST, but not a controlprotein (MBP) when immobilized on TentaGel. Furthermore, unlabeled GSTwas retained by the TentaGel-peptoid beads in the presence of a1000-fold excess of E. coli extract (FIG. 20B). As shown in FIG. 20C,this was the case using GST concentrations of 1 μM to 100 nM. When theprotein concentration was 10 nM, little or no fluorescence abovebackground was observed (not shown). Finally, solution binding studieswere performed employing isothermal titration calorimetry (ITC)resulting in an equilibrium dissociation constant of 62 μM for thepeptoid/protein complex.

Example 5 Chimeric Binding Element Studies

Semi-Automated Screening of a Library. Stoll et al. previously reportedthat the chalcone general formula 1 (FIG. 22) associates with thep53-binding domain of the proto-oncoprotein Mdm2 weakly (K_(D)=220 μM)(Stoll et al., 2001). A structural model derived from NMR data (Stoll etal., 2001) suggested that the carboxylate group of the chalcone wasoriented away from the protein and could be utilized for attachment toother moieties. The inventors employed chalcone (i.e., as a firstbinding element of a chimeric binding element) as a test case for ascreening strategy to identify chimeric binding elements for use incompositions and methods of the present invention. A combinatoriallibrary of peptoids (Figliozzi et al., 1996; Kirshenbaum et al., 1998;Burkoth et al., 2002) was synthesized by split and pool solid phasesynthesis (Lam et al., 1991) using the five amines pictured in FIG. 22.This library contains 78,125 (5⁷) different compounds. The library hasseven positions that were randomized completely, a central Nser (seeFIG. 22 for nomenclature) unit and two constant Npip residues betweenthe library and the polyethylene glycol layer that coats the surface ofthe Tentagel bead. The Npip-Npip linker was included to facilitateeventual identification of hits since these are identified by Edmandegradation and the quality of the sequence falls off drastically closeto the polyethylene glycol layer. Enough beads were employed in thesplit and pool chemistry that each compound should be representedapproximately ten times in the library.

To make a chalcone-capped library, approximately 10% (=78,000 beads) ofthe aforementioned library (with side chain protecting groups intact)was capped at its N-terminal end with a lysine residue modified withchalcone formula 1 via a side chain amide bond (FIG. 22). The protectinggroups were then removed to provide the desired library ready forscreening.

Approximately 78,000 beads was pre-sorted using a fluorescent beadsorter (COPAS SELECT 500 from Union Biometrica, Inc., Somerville, Mass.)to remove beads that exhibited intense autofluorescence. The remaining66,862 beads were then incubated with fluorescein-labeled MBP-Mdm2protein under conditions that were too demanding for the chalcone aloneto support detectable binding of the labeled protein to a bead (500 nMMBP-Mdm2+10,000-fold excess of E. coli lysate in TBST+1M NaCl+1%Tween-20). After a two-hour incubation followed by thorough washing, thebeads were then poured into the COPAS instrument and sorted byfluorescence intensity. Only four beads (0.00598% of the library)exhibited fluorescent well above the background. These were collected bythe sorter. This low hit rate suggested that the conditions employed hadindeed selected stringently for the best ligands in the library. Tovalidate that the instrument had identified the appropriate beads, oneof the “hits” was placed on a microscope slide along with several of the“negative” beads from the sort. A fluorescence micrograph of part ofthis field is shown in FIG. 23. The identity of the compounds on each ofthe four beads was determined by automated Edman sequencing. Two of thefour were identical, having the structure:NH₂-Lys(Chalcone)-Nlys-Npip-Nlys-Nser-Nlys-Nlys-Nlys-Nlys-Npip-Npip(residues that were varied in the library are underlined) (FIG. 23). Theother two hits(NH₂-Lys(Chalcone)-Npip-Nser-Nlys-Nser-Npip-Nlys-Nlys-Nlys-Npip-Npip andNH₂-Lys(Chalcone)-Nlys-Nlys-Nbsa-Nser-Nall-Nlys-Nlys-Nlys-Npip-Npip,were also Nlys-rich, indicating that a highly basic peptoid facilitateshigher affinity binding to the target protein.

Binding studies confirm high affinity Mdm2/MECA binding.NH₂-Lys(Chalcone)-Nlys-Npip-Nlys-Nser-Nlys-Nlys-Nlys-Nlys-Npip-Npip(formula 2) was resynthesized and purified by reverse-phase HPLC.Titration experiments using MBP-Mdm2 monitored by isothermal calorimetry(ITC) revealed a solution equilibrium dissociation constant of 1.3(±0.4) W. The K_(D) of the chalcone formula 1/MBP-Mdm2 complex was notable to be determined under the same conditions by ITC due toinsufficient solubility of the small molecule. However, under conditionsused by the inventor, the K_(D) must be at least the reported 220 μM orhigher. Therefore, the ITC data indicate an improvement of at least170-fold in the affinity of the chalcone-peptoid chimera formula 2relative to the parent compound formula 1. Interestingly, the K_(D) ofthe complex of MBP-Mdm2 and the peptoidNH₂-Nlys-Npip-Nlys-Nser-Nlys-Nlys-Nlys-Nlys-Npip-Npip lacking thechalcone cap was poor (378 μM, FIG. 24B), demonstrating that neitherpiece of the binding element is itself a high affinity capture agent. AnITC experiment was also done with chimera formula 2 and MBP lacking theMdm2 fusion. Only the heat of dilution of the titrant was observed inthis experiment (FIG. 24C), demonstrating little or no binding (K_(D)mM). These data demonstrate that interactions between formula 2 and MBPcontribute little or nothing to the observed binding affinity and thatthe MECA derived from the chimeric binding element is specific for Mdm2.

It is also interesting to note that the same library employed above, butlacking the Chalcone-Lys cap, was screened against MBP-Mdm2 under lessdemanding conditions and a completely different set of peptoid sequenceswas isolated, strengthening the idea that the binding element of formula2 is a unique species that is greater than the sum of its parts.

To probe the binding chemistry of chimeric binding ligand formula 2 on asolid support, which is more relevant to the issue of creating highaffinity protein capture agents, TentaGel beads were prepared thatdisplay either chalcone formula 1 alone, the 10mer peptoidNH₂-Nlys-Npip-Nlys-Nser-Nlys-Nlys-Nlys-Nlys-Npip-Npip alone, or thechalcone-peptoid chimeric binding element of formula 2. In theexperiment shown in FIG. 24D, the beads and the Texas Red-labeledproteins (100 nM) indicated in the figure were incubated under demandingbuffer conditions (1M NaCl and 1% Tween-20 in the presence of a 100-foldexcess of E. coli proteins), then washed thoroughly. Two populations ofbeads were then mixed in a 1:1 ratio and photographed under afluorescence microscope to provide a direct comparison. FIG. 24D andFIG. 24E show the contrast between the solid phase MBP-Mdm2-bindingaffinity of the chalcone-peptoid formula 2 and the peptoid alone (FIG.24D) and chalcone formula 1 alone (FIG. 24E), respectively. In each caseone set of beads is much brighter than the other and subsequent Edmansequencing of the bright and dark beads confirmed that in both cases thebright beads displayed chimeric binding element formula 2. FIG. 24Fshows the high level of contrast between the chalcone-peptoid-displayingbeads that had been incubated with either labeled MBP-Mdm2 or MBP, againdemonstrating specificity. These data agree qualitatively with the ITCresults in that they show the chalcone-peptoid chimera has a higheraffinity for Mdm2 than does either individual component of the chimera.

To better judge the apparent affinity of the immobilizedchalcone-peptide chimera formula 2 for labeled Mdm2, the study shown inFIG. 25 was conducted. In this case, a more typical biochemical buffer(150 mM NaCl and 0.1% detergent) was employed and the indicatedconcentration of labeled Mdm2 was mixed with 100-fold excess of E. coliproteins. After thorough washing, the beads were photographed in thefluorescence microscope using identical settings in each case. As can beseen in FIG. 24, capture of Mdm2 was apparent down to a proteinconcentration of 10 nM. The image at 1 nM Mdm2 was similar to that of acontrol bead displaying a different ligand.

A high affinity ubiquitin capture agent. For many proteins, even modestaffinity lead compounds are not available. Therefore to evaluate achimeric binding element from scratch, a high-affinity ubiquitin captureagent was generated by a two-step screening process in which a naïvepeptide library was first screened under relatively mild conditions andthen a hit from this screen was used to cap a chimeric binding elementlibrary that was then screened under more demanding conditions.

In the first round a library of seven residue peptides was screened,resulting in the isolation of the peptide NH₂-RWDRYYF. Titrationexperiments monitored by ITC revealed a K_(D) of 33 (±5) μM for thepeptide/ubiquitin complex (FIG. 26A). A new peptide library was thenconstructed on TentaGel beads by split and pool synthesis of the formNH₂-X₇-S-RWDRYYF, where X represents a randomized position using theamino acids A, E, G, H, K, L, N, R, T, or W. A fraction of this library(≈250,000 beads) was then incubated with fluorescently-labeled ubiquitin(200 nM) in the presence of a 10,000-fold excess of unlabeled proteinsin a buffer containing 0.5 M NaCl and 0.5% Tween-20 detergent. Inaddition, a 1000-fold excess of synthetic lead peptide (NH₂—RWDRYYF) wasalso included in the buffer to block capture of ubiquitin by moleculesthat represent only a modest improvement over the lead peptide. Underthese conditions, only three beads (0.0012% of the library) fluorescedwell above background. The structure of the peptides was deduced byEdman sequencing. One of them (NH₂-WGLRALESRWDRYYF) was resynthesizedand purified. ITC experiments revealed that the chimeric binding elementexhibited only a slight improvement over the lead in terms of itssolution affinity for ubiquitin (FIG. 26A; K_(D)=12 (±4) μM).

To study the behavior of the ubiquitin-targeted chimeric binding elementin capture assays, the peptide was synthesized on Tentagel and employedin “pull-down” assays. Beads displaying NH₂-WGLRALESRWDRYYF, a controlpeptide NH₂-HHRSHYKSMPRFMDYWEDL, or no peptide at all, were incubatedwith the indicated concentration of unlabeled ubiquitin in the presenceof a 1000-fold excess of E. coli proteins (FIG. 26B). After thoroughwashing, the beads were then probed with Texas Red-labeledanti-ubiquitin-labeled polyclonal antibodies to visualize the boundprotein. As shown in FIG. 26B, binding of ubiquitin by the chimericbinding element was very strong at 4 nM ubiquitin and easily detectableeven at 0.8 nM ubiquitin. In all cases, the level of fluorescence fromthe chimeric binding element-displaying beads was much higher than fromeither set of control beads (see FIG. 26C for a direct comparison).Moreover, when the experiment was repeated with ubiquitin omitted fromthe solution, all of the beads exhibited the same low-level backgroundsignal (data not shown). While these binding assays are onlysemi-quantitative in nature, the data indicate that the functionaldissociation constant of the immobilized chimeric binding element forubiquitin is at least in the low nanomolar range.

Capture agents compared to solution ligands. The apparent bindingaffinity of the chimeric binding elements were considerably better whenthese compounds were immobilized than was the case free in solution. Ofcourse, the bead-binding assays employed here are only semi-quantitativeand in any case, one cannot compare apparent affinities to true solutionK_(D)s rigorously. Furthermore, it is common to observe enhanced bindingof a soluble analyte to a resin-bound compound since once the targetmolecule is bound, it finds itself in a local environment of very highligand concentration, making escape from the environment of the beadunlikely. It may be that this effect can explain much of the apparentdifferences in binding affinity between the solution and immobilizedMdm2 chimeric binding element. Protein-binding compounds isolated fromnaive (i.e., non-chimeric binding element) libraries of peptides orpeptoids generally form complexes with solution K_(D)s in the low to midμM range. But when binding of the labeled target protein to theTentaGel-immobilized binding element is examined, binding can bedetected routinely at a soluble protein concentration 10-100-fold lowerthan the solution K_(D) value (unpublished results), even in cases wheremultivalent contacts are not possible. However, it seems unlikely thatthis mechanism could account completely for the much larger differencebetween these values observed in the ubiquitin study (solution K_(D) ofapproximately 12 but efficient capture at or below 1 nM). Another cleardifference between the two chimeric binding elements is that theMdm2-binding molecule formula 2 shows a large enhancement in itssolution affinity over the lead chalcone formula 1, whereas theubiquitin-binding chimeric binding element is only 3-fold better insolution than the lead peptide. Thus, a more likely explanation is thatthe immobilized ubiquitin-binding peptide NH₂-WGLRALESRWDRYYF bindsubiquitin tightly because two different surface-bound moleculescollaborate in an avidity-based event. Specifically, the lead peptidefrom one chimeric binding element molecule and the library-derivedsegment from another converge on a single molecule of ubiquitin. This isnot surprising since the screen was carried out with resin-immobilizedmolecules and in fact is not a problem if the chimeric binding elementswill be employed as immobilized protein capture agents.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 4,946,778-   U.S. Pat. No. 5,582,981-   U.S. Pat. No. 5,617,060-   U.S. Pat. No. 5,719,060-   U.S. Pat. No. 5,756,291-   U.S. Pat. No. 5,780,610-   U.S. Pat. No. 5,792,613-   U.S. Pat. No. 5,840,867-   U.S. Pat. No. 6,225,047-   U.S. Pat. No. 6,329,209-   U.S. Pat. No. 6,344,334-   U.S. Pat. No. 6,461,515-   U.S. Pat. No. 6,475,391-   Alberts et al., In: Molecular Biology of the Cell, 3rd ed., Garland    Publishing, Inc. NY, 1994.-   Bachhawat-Sikder and Kodadek, J. Amer. Chem. Soc., 125:9550-9551,    2003.-   Barany and Merrifield, In: The Peptides, Gross and Meienhofer    (Eds.), Academic Press, NY, 1-284, 1979.-   Blackwell et al., Chem. Biol., 8:1167-1182, 2001.-   Boeijen and Liskamp, J. Tetrahedron Lett., 39:3589-3592, 1998.-   Bottger et al., Curr. Biol., 7:860-869, 1997.-   Brocchini et al., J. Am. Chem. Soc., 119:4553-4554, 1997.-   Burkoth et al., Chem. Biol., 9:647-654, 2002.-   Chene et al., J. Mol. Biol., 299:245-253, 2000.-   Cho et al., Bioorg Med Chem 7, 1171-1179, 1999.-   Clemons et al., Chem. Biol., 8:1183-1195, 2001.-   Colas et al., Nature, 380:548-550, 1996.-   Cole et al., In: Monoclonal Antibodies and Cancer Therapy, Alan R.    Liss, Inc., 77-96, 1985.-   Cussac et al., FASEB J., 13:31-38, 1999.-   Eggers et al., Biotechniques, 17:516-525, 1994.-   Eichler et al., Medicinal Research Reviews 15, 481-496, 1995.-   Fairbrother et al., Biochemistry, 37:17754-17764, 1998.-   Famlouk and Jenne, Curr. Opin. Chem. Biol., 2:320-327, 1998.-   Fancy and Kodadek, Proc. Natl. Acad. Sci. USA, 96:6020-6024, 1999.-   Figliozzi et al., Methods Enzymol., 267:437-447, 1996.-   Gallop et al., J. Med. Chem., 37(9):1233-1251, 1994.-   Gordon et al., J. Med. Chem., 37(10):1385-401, 1994.-   Haab et al., Genome Biol., 2(2):RESEARCH0004, 2001.-   Han and Kodadek, J. Biol. Chem., 275:4979-14984, 2000.-   Heyduk, et al., Method Enzymol. 274:492-503, 1996.-   Hornak, In: The Basics of MRI, 2002.-   Huang et al., Anal. Biochem., 294:55-62, 2001.-   Huang, J. Immunol. Methods, 255:1-13, 2001.-   Hung et al. Cancer Res., 62:2806-2812, 2002.-   Huse et al., Science, 246:1275-1281, 1989.-   Jhaveri et al., Nature Biotechnol., 18:1293-1297, 2000.-   Johnston et al., Cell, 50:143-146, 1987.-   Kanemitsu, Comb Chem High Throughput Screen, 5(5):339-360, 2002.-   Kiessling et al., Curr. Opin. Chem. Biol., 4:696-703, 2000.-   Kirshenbaum et al., Proc. Natl. Acad. Sci. USA, 95:4303-4308, 1998.-   Kitov et al., Nature, 403:669-672, 2000.-   Kitov et al., J. Amer. Chem. Soc., 125:3284-3294, 2003.-   Kodadek, Chem. Biol., 8:105-115, 2001.-   Kodadek, Trends Biochem. Sci., 27(6):295-300, 2002.-   Koehler et al., J. Amer. Chem. Soc., 125:8420-8421, 2003.-   Kohler and Milstein, Nature, 256:495-497, 1975.-   Kozbor et al., Immunology Today, 4:72, 1983.-   Kuruvilla et al., Nature, 416:653-657, 2002.-   Kussie et al., Science, 2(74):948-953, 1996.-   Lam et al., Nature, 354:82-84, 1991.-   Leavitt and Freire, Curr. Opin. Struct. Biol., 11:560-566, 2001.-   LePlae et al., J. Amer. Chem. Soc., 124:6820-6821, 2002.-   Lodish et al., In: Molecular Cell Biology, 4th ed., W.H. Freeman and    Company, 2000.-   Maly et al., Proc. Natl. Acad. Sci. USA, 97:2419-2424, 2000.-   Melcher and Xu, EMBO J., 20:841-851, 2001.-   Merrifield, Science, 232(4748):341-347, 1986.-   Merritt et al., J. Amer. Chem, Soc., 124:8818-8824, 2002.-   Needels et al., Proc. Natl. Acad. Sci. USA, 90:10700-10704, 1993.-   Olejniczak et al., J. Amer. Chem. Soc., 119:5828-5832, 1997.-   Oliver et al., Clinical Chemistry, 44:2053-2060, 2000.-   Olivos et al., Org. Lett., 4:4057-4059, 2002.-   Osborne et al., Curr. Opin. Chem. Biol., 1:5-9, 1997.-   Ostergaard and Holm, Mol. Divers., 3:17-27, 1997.-   PCT Appln. WO 00/56934-   PCT Appln. WO 98/59360-   PCT Appln. WO 99/51773-   PCT Appln. WO98/59360-   Pons et al., Eur. J. Org. Chem., 853-859, 1998.-   Radhakrishnan et al., Cell, 91:741-752, 1997.-   Roberts and Szostak, Proc. Natl. Acad. Sci. USA, 94:12297-12302,    1997.-   Schreiber, Chem. Eng. News, 81:51-61, 2003.-   Schultz et al., Cytometry, 43:239-247, 2001.-   Seethsnunan et al., Nature Biotechnol., 19:336-341, 2001.-   Shukery et al., Science, 274, 1531-1534, 1996.-   Sternsdorf et al., J Biol Chem. 274(18):12555-66, 1999.-   Stewart and Young, In: Solid Phase Peptide Synthesis, 2d. ed.,    Pierce Chemical Co., 1984.-   Stoll et al., Biochemistry, 40:336-344, 2001.-   Tam et al., J. Am. Chem. Soc., 105:6442, 1983.-   Terskikh et al., Proc. Natl. Acad. Sci. USA, 94:1663-1668, 1997.-   Thompson and Ellman, Chem. Rev., 96(1):555-600, 1996.-   Thom et al., J. Amer. Chem. Soc., 123:10113-10114, 2001.-   Uno et al., Tetrahedron Lett., 40:1475-1478, 1999.-   Vaish et al., Nature Biotech., 20:810-815, 2002.-   Vignali, J. of Immunol. Methods, 243:243-255, 2000.-   Walter et al., Curr. Opin. Microbiol., 3:298-302, 2000.-   Wender et al., Proc. Natl. Acad. Sci. USA, 97:13003-13008, 2000.-   Wiese et al., Clinical Chemistry, 47:1451-1457, 2001.-   Wilson et al., Proc. Natl. Acad. Sci. USA, 98:3750-3755, 2001.-   Yang et al., J. Amer. Chem. Soc., 121:589-590, 1999.-   Zuckerman et al., J. Med. Chem., 37:2678-2685, 1994.

1. A composition for assessing the presence of at least a first targetmolecule in a sample comprising a plurality of low-to-moderate affinitybinding elements distributed on a surface of, and operatively coupled toa support, wherein concomitant binding of the first target molecule totwo or more of the binding elements results in a high affinityinteraction with the first target molecule.
 2. The composition of claim1, wherein the binding elements are peptides, peptoids (N-substitutedoligoglycines) or other peptide-like oligomers.
 3. The composition ofclaim 1, wherein the plurality of binding elements comprises at least afirst and a second binding element having distinct binding specificityfor a target molecule as compared to each other.
 4. The composition ofclaim 1, wherein a first binding element is operatively coupled to thesecond binding element.
 5. The composition of claim 4, wherein a spaceris operatively coupled to the first binding element, the second bindingelement or both the first and second binding element.
 6. The compositionof claim 5, wherein the second binding element is an oligomer.
 7. Thecomposition of claim 6, wherein the oligomer is a peptide or peptidederivative.
 8. The composition of claim 7, wherein the peptidederivative is comprised of one or more non-natural amino acid.
 9. Thecomposition of claim 7, wherein the peptide derivative comprises one ormore peptoid monomers. 10.-14. (canceled)
 15. The composition of claim1, wherein the sample is an environmental sample, a cell lysate, a bloodsample, a sputum sample or a urine sample. 16.-20. (canceled)
 21. Thecomposition of claim 1, wherein the binding elements are distributedrandomly on the surface of the support.
 22. The composition of claim 1,further comprising at least a third and a fourth low-to-moderate bindingelement that bind a second target molecule, the third and fourth bindingelement distributed on a surface of and operatively coupled to, thesupport, wherein concomitant binding of the second target molecule tothe third and fourth binding elements results in a high affinityinteraction with the second target molecule. 23.-27. (canceled)
 28. Amethod of determining the presence of a target molecule in a samplecomprising: a) exposing the sample to a plurality of low-to-moderateaffinity binding elements distributed on a surface of, and operativelycoupled to a support, wherein concomitant binding of the target moleculeto at least a two of the binding elements results in a specific highaffinity interaction with the target molecule; and b) evaluating bindingof the target molecule to the binding elements. 29.-35. (canceled)
 36. Amethod of producing a chimeric binding element comprising: a) providinga first low-to-moderate affinity binding element; b) providing acombinatorial library of oligomers; c) operatively coupling the firstbinding element to one or more members of the combinatorial library; andd) identifying a first binding element/oligomer combination with a highaffinity for a target molecule, wherein at least a portion of theoligomer is a second binding element. 37.-38. (canceled)
 39. The methodof claim 36, wherein the peptide derivative comprises one or morepeptoid monomers. 40.-43. (canceled)